Processing reads, mapping, and building a count matrix

baseDir <- "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette"
library(stringr)
library(CLIPflexR)
library(SummarizedExperiment)
library(GenomicRanges)
library(rtracklayer)

Process standard/BrdU-CLIP libraries using CLIPflexR

This is an alternative to the CTK processing pipeline for CLIP data, and returns a peak matrix with genomic locations of peaks and counts in each peak by sample

Initial processing and quality control

Decompress fastq files

Use compressed fastq files as input; R-wrapped decompression function using bzip2.
CLIPflexR documentation

#define the path to your compressed fastq file(s)
#here we have one example fastq.gz generated by standard CLIP (linker ligations; KRG092419_S1_L001_R1_001.fastq.gz)
#and one example fastq.gz generated by BrdU-CLIP (KRG091819_S1_L001_R1_001.fastq.gz)
testFQ <- dir(baseDir, pattern = ".gz", full.names = TRUE)
testFQ

## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001.fastq.gz"
## [2] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001.fastq.gz"

FqFile <- lapply(testFQ, decompress, overwrite=TRUE)
FqFile

## [[1]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001.fastq"
## 
## [[2]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001.fastq"

You can look at the quality of your data (wrapping FASTX’s quality stats)

Get quality statistics from your run using R-wrapped fastx_quality_stats from FASTX_toolkit.
CLIPflexR documentation

#defaults are set for illumina quality encoding, minimum quality score of 20 for 80% of bases
stats <- lapply(FqFile, fastx_quality_stats)
stats

## [[1]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001.txt"
## 
## [[2]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001.txt"

stats <- read.delim(stats[[1]],header = T, sep = "\t")
head(stats)

##   column    count min max       sum  mean Q1 med Q3 IQR lW rW A_Count
## 1      1 22052373  16  36 712698638 32.32 32  33 34   2 29 36 7104877
## 2      2 22052373   2  36 696285989 31.57 32  33 34   2 29 36 6911254
## 3      3 22052373  16  36 717065818 32.52 32  33 34   2 29 36 7053127
## 4      4 22052373  16  36 718409762 32.58 32  33 34   2 29 36 7049046
## 5      5 22052373  16  36 715824000 32.46 32  33 34   2 29 36 7020505
## 6      6 22052373  14  37 775710593 35.18 34  37 37   3 30 37 6997082
##   C_Count G_Count T_Count N_Count Max_count
## 1 4814492 3873005 6259999       0  22052373
## 2 4622185 3607472 6192199  719263  22052373
## 3 5082531 3618036 6298679       0  22052373
## 4 5060538 3650501 6292288       0  22052373
## 5 5121322 3657985 6252561       0  22052373
## 6 5109816 3666739 6278736       0  22052373

Quality filter (wrapping FastX’s quality filter)

Filtering fastq files using R-wrapped fastq_quality_filter from FASTX_toolkit.
qEncoding: Quality encoding, default = 33 (corresponds to Illumina and Sanger quality scores); minimumQuality: minimum quality score to keep, default = 20. Only keep reads with quality score greater than this threshold. minimumPercentOfRead: minimum percent of bases that must have [-q] minimumQuality.
CLIPflexR documentation

FqFile_QF <- lapply(FqFile, fastq_quality_filter)
FqFile_QF

## [[1]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/QF_KRG091819_S1_L001_R1_001.fastq"
## 
## [[2]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/QF_KRG092419_S1_L001_R1_001.fastq"

Fastx_collapser (wrapping FASTX’s fastx_collapser)

Collapse duplicate sequences into unique reads using R-wrapped fastx_collapser from FASTX_toolkit.
qEncoding: Quality encoding, default = 33 (corresponds to Illumina and Sanger quality scores).
CLIPflexR documentation

coll_FqFile_QF <- lapply(FqFile_QF, fastx_collapser)
coll_FqFile_QF

## [[1]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/QF_KRG091819_S1_L001_R1_001_collapse.fasta"
## 
## [[2]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/QF_KRG092419_S1_L001_R1_001_collapse.fasta"

Follow these steps to process standard CLIP libraries

De-multiplex samples (wrapping FASTX’s barcode splitter)

De-multiplex samples by indices using R-wrapped fastx_barcode_splitter from FASTX_toolkit.
mismatches: number of mismatches allowed in barcode sequence, 0 (default); bcFile: barcode file, see example below.
CLIPflexR documentation

#provide the path to your barcode file,  which must contain one sample name and its corresponding index sequence for  each line, and be tab delimited

#For example:
#Aag2_rIgG_Exp6A CGATG
#Aag2_rIgG_Exp6B TTAGG

BCfile <- dir(baseDir, pattern="BC",  full.names = TRUE)
BCfile <- grep("stats", BCfile, value =T, invert = T)
BCfile

## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_BC.txt"
## [2] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_BC.txt"

#take standard libraries only
samples_split_std <- fastx_barcode_splitter(coll_FqFile_QF[[2]], bcFile = BCfile[[2]], verbose = TRUE)

## barcode_splitter command is cat /Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/QF_KRG092419_S1_L001_R1_001_collapse.fasta | /Users/kathryn/Library/r-miniconda/envs/CLIPflexR_0.1.19/bin/fastx_barcode_splitter.pl --bcfile /Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_BC.txt --bol --mismatches 0 --prefix '/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001_collapse_'

## barcode_splitter arguments are  --bcfile /Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_BC.txt--bol--mismatches 0 --prefix '/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001_collapse_'

#files will be written with a prefix appended for the run, and then the sample name given in the barcode file
#split samples will not have an extension but will be in FASTA format
samples_split_std

##  [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001_collapse_Aag2_Ago1_Exp7A"   
##  [2] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001_collapse_Aag2_Ago1_Exp7B"   
##  [3] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001_collapse_Aag2_Ago2_Exp7A"   
##  [4] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001_collapse_Aag2_Ago2_Exp7B"   
##  [5] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001_collapse_Aag2_mIgG_Exp7A"   
##  [6] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001_collapse_Aag2_mIgG_Exp7B"   
##  [7] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001_collapse_Aag2_rIgG_Exp7A"   
##  [8] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001_collapse_Aag2_rIgG_Exp7B"   
##  [9] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001_collapse_aegypti_Ago2_Exp7A"
## [10] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001_collapse_aegypti_Ago2_Exp7B"
## [11] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001_collapse_aegypti_mIgG_Exp7A"
## [12] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001_collapse_aegypti_mIgG_Exp7B"
## [13] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001_collapse_aegypti_mIgG_Exp7C"
## [14] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001_collapse_aegypti_mIgG_Exp7D"

#sample_stats will also be written, and contain how many reads were extracted for each sample and the file name for that run ID
std_samples_stats <- read.delim(paste0(baseDir, "/KRG092419_BC_stats.txt"), header = T, sep =  "\t")
head(std_samples_stats)

##           Barcode  Count
## 1 Aag2_Ago1_Exp7A 612767
## 2 Aag2_Ago1_Exp7B 579807
## 3 Aag2_Ago2_Exp7A 894236
## 4 Aag2_Ago2_Exp7B 701068
## 5 Aag2_mIgG_Exp7A 310822
## 6 Aag2_mIgG_Exp7B  46491
##                                                                                                                 Location
## 1 /Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001_collapse_Aag2_Ago1_Exp7A
## 2 /Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001_collapse_Aag2_Ago1_Exp7B
## 3 /Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001_collapse_Aag2_Ago2_Exp7A
## 4 /Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001_collapse_Aag2_Ago2_Exp7B
## 5 /Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001_collapse_Aag2_mIgG_Exp7A
## 6 /Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001_collapse_Aag2_mIgG_Exp7B

Now remove the 5’ index, linker, and random barcode sequences (wrapping CTK’s stripBarcode.pl)

Strip random barcodes/linkers with specific length from reads using R-wrapped stripBarcode.pl from CTK.
linkerlength: barcode length; inputformat: format of input sequences, fasta (default).
CLIPflexR documentation

samples_split_std_rm5 <- lapply(samples_split_std, ctk_stripBarcode, linkerlength = 27, inputFormat="fasta")
samples_split_std_rm5

## [[1]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001_collapse_Aag2_Ago1_Exp7A_rm5.fa"
## 
## [[2]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001_collapse_Aag2_Ago1_Exp7B_rm5.fa"
## 
## [[3]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001_collapse_Aag2_Ago2_Exp7A_rm5.fa"
## 
## [[4]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001_collapse_Aag2_Ago2_Exp7B_rm5.fa"
## 
## [[5]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001_collapse_Aag2_mIgG_Exp7A_rm5.fa"
## 
## [[6]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001_collapse_Aag2_mIgG_Exp7B_rm5.fa"
## 
## [[7]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001_collapse_Aag2_rIgG_Exp7A_rm5.fa"
## 
## [[8]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001_collapse_Aag2_rIgG_Exp7B_rm5.fa"
## 
## [[9]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001_collapse_aegypti_Ago2_Exp7A_rm5.fa"
## 
## [[10]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001_collapse_aegypti_Ago2_Exp7B_rm5.fa"
## 
## [[11]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001_collapse_aegypti_mIgG_Exp7A_rm5.fa"
## 
## [[12]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001_collapse_aegypti_mIgG_Exp7B_rm5.fa"
## 
## [[13]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001_collapse_aegypti_mIgG_Exp7C_rm5.fa"
## 
## [[14]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001_collapse_aegypti_mIgG_Exp7D_rm5.fa"

Now remove the 3’ linker (wrapping FASTX’s clipper)

Trimming 3’-end adapter sequences using R-wrapped fastx_clipper function from FASTX_toolkit.
adapter: clip this sequence from the 3’-end of reads, “GTGTCAGTCACTTCCAGCGG” (default); length: minimum read length, only keep reads with length equal or greater then this threshold, 18 (default).
CLIPflexR documentation

samples_split_std_rm5_rm3 <- lapply(samples_split_std_rm5, fastx_clipper, length = 18, adapter="GTGTCAGTCACTTCCAGCGG")
samples_split_std_rm5_rm3

## [[1]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001_collapse_Aag2_Ago1_Exp7A_rm5_clip.fa"
## 
## [[2]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001_collapse_Aag2_Ago1_Exp7B_rm5_clip.fa"
## 
## [[3]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001_collapse_Aag2_Ago2_Exp7A_rm5_clip.fa"
## 
## [[4]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001_collapse_Aag2_Ago2_Exp7B_rm5_clip.fa"
## 
## [[5]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001_collapse_Aag2_mIgG_Exp7A_rm5_clip.fa"
## 
## [[6]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001_collapse_Aag2_mIgG_Exp7B_rm5_clip.fa"
## 
## [[7]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001_collapse_Aag2_rIgG_Exp7A_rm5_clip.fa"
## 
## [[8]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001_collapse_Aag2_rIgG_Exp7B_rm5_clip.fa"
## 
## [[9]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001_collapse_aegypti_Ago2_Exp7A_rm5_clip.fa"
## 
## [[10]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001_collapse_aegypti_Ago2_Exp7B_rm5_clip.fa"
## 
## [[11]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001_collapse_aegypti_mIgG_Exp7A_rm5_clip.fa"
## 
## [[12]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001_collapse_aegypti_mIgG_Exp7B_rm5_clip.fa"
## 
## [[13]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001_collapse_aegypti_mIgG_Exp7C_rm5_clip.fa"
## 
## [[14]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001_collapse_aegypti_mIgG_Exp7D_rm5_clip.fa"

These reads are now ready to map to your genome

Follow these steps to process BrdU-CLIP libraries

First strip random barcode sequences (UMI) from the 5’ end of reads (wrapping CTK’s stripBarcode.pl)

BrdU_rm5 <- ctk_stripBarcode(coll_FqFile_QF[[1]], linkerlength = 7, inputFormat="fasta")
BrdU_rm5

## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/QF_KRG091819_S1_L001_R1_001_collapse_rm5.fasta"

De-multiplex samples (wrapping FASTX’s barcode splitter)

samples_split_BrdU <- fastx_barcode_splitter(BrdU_rm5, bcFile = BCfile[[1]], verbose = TRUE)

## barcode_splitter command is cat /Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/QF_KRG091819_S1_L001_R1_001_collapse_rm5.fasta | /Users/kathryn/Library/r-miniconda/envs/CLIPflexR_0.1.19/bin/fastx_barcode_splitter.pl --bcfile /Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_BC.txt --bol --mismatches 0 --prefix '/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_'

## barcode_splitter arguments are  --bcfile /Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_BC.txt--bol--mismatches 0 --prefix '/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_'

#files will be written with a prefix appended for the run, and then the sample name given in the barcode file
#split samples will not have an extension but will be in FASTA format
samples_split_BrdU

##  [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_Aag2_Ago1_Exp6A"   
##  [2] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_Aag2_Ago1_Exp6B"   
##  [3] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_Aag2_Ago2_Exp6A"   
##  [4] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_Aag2_Ago2_Exp6B"   
##  [5] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_Aag2_mIgG_Exp6A"   
##  [6] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_Aag2_mIgG_Exp6B"   
##  [7] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_Aag2_rIgG_Exp6A"   
##  [8] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_Aag2_rIgG_Exp6B"   
##  [9] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_aegypti_Ago1_Exp6A"
## [10] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_aegypti_Ago1_Exp6B"
## [11] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_aegypti_Ago2_Exp6A"
## [12] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_aegypti_Ago2_Exp6B"
## [13] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_aegypti_mIgG_Exp6A"
## [14] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_aegypti_mIgG_Exp6B"
## [15] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_aegypti_rIgG_Exp6A"
## [16] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_aegypti_rIgG_Exp6B"

#sample_stats will also be written, and contain how many reads were extracted for each sample and the file name for that run ID
BrdU_samples_stats <- read.delim(paste0(baseDir, "/KRG091819_BC_stats.txt"), header = T, sep =  "\t")
head(BrdU_samples_stats)

##           Barcode  Count
## 1 Aag2_Ago1_Exp6A 491943
## 2 Aag2_Ago1_Exp6B 507596
## 3 Aag2_Ago2_Exp6A 673944
## 4 Aag2_Ago2_Exp6B 393337
## 5 Aag2_mIgG_Exp6A   1688
## 6 Aag2_mIgG_Exp6B    926
##                                                                                                                     Location
## 1 /Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_Aag2_Ago1_Exp6A
## 2 /Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_Aag2_Ago1_Exp6B
## 3 /Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_Aag2_Ago2_Exp6A
## 4 /Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_Aag2_Ago2_Exp6B
## 5 /Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_Aag2_mIgG_Exp6A
## 6 /Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_Aag2_mIgG_Exp6B

Trim 3 “not G” (DDD) and index (6nt) from the 5’ end of reads (wrapping FASTX’s fastx_trimmer)

Trimming 5’-end adapter sequences using R-wrapped fastx_trimmer function from FASTX_toolkit.
read_start: read starts at this base, 10 (default); read_end: read ends at this base, NULL (default).
CLIPflexR documentation

BrdU_split_rm5 <- lapply(samples_split_BrdU, fastx_trimmer, read_start = 10)
BrdU_split_rm5

## [[1]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_Aag2_Ago1_Exp6A_trim.fa"
## 
## [[2]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_Aag2_Ago1_Exp6B_trim.fa"
## 
## [[3]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_Aag2_Ago2_Exp6A_trim.fa"
## 
## [[4]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_Aag2_Ago2_Exp6B_trim.fa"
## 
## [[5]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_Aag2_mIgG_Exp6A_trim.fa"
## 
## [[6]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_Aag2_mIgG_Exp6B_trim.fa"
## 
## [[7]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_Aag2_rIgG_Exp6A_trim.fa"
## 
## [[8]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_Aag2_rIgG_Exp6B_trim.fa"
## 
## [[9]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_aegypti_Ago1_Exp6A_trim.fa"
## 
## [[10]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_aegypti_Ago1_Exp6B_trim.fa"
## 
## [[11]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_aegypti_Ago2_Exp6A_trim.fa"
## 
## [[12]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_aegypti_Ago2_Exp6B_trim.fa"
## 
## [[13]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_aegypti_mIgG_Exp6A_trim.fa"
## 
## [[14]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_aegypti_mIgG_Exp6B_trim.fa"
## 
## [[15]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_aegypti_rIgG_Exp6A_trim.fa"
## 
## [[16]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_aegypti_rIgG_Exp6B_trim.fa"

Fastx_clipper (wrapping FASTX’s fastx_clipper)

BrdU_split_rm5_rm3 <- lapply(BrdU_split_rm5, fastx_clipper, length = 18, adapter="GTGTCAGTCACTTCCAGCGG")
BrdU_split_rm5_rm3

## [[1]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_Aag2_Ago1_Exp6A_trim_clip.fa"
## 
## [[2]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_Aag2_Ago1_Exp6B_trim_clip.fa"
## 
## [[3]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_Aag2_Ago2_Exp6A_trim_clip.fa"
## 
## [[4]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_Aag2_Ago2_Exp6B_trim_clip.fa"
## 
## [[5]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_Aag2_mIgG_Exp6A_trim_clip.fa"
## 
## [[6]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_Aag2_mIgG_Exp6B_trim_clip.fa"
## 
## [[7]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_Aag2_rIgG_Exp6A_trim_clip.fa"
## 
## [[8]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_Aag2_rIgG_Exp6B_trim_clip.fa"
## 
## [[9]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_aegypti_Ago1_Exp6A_trim_clip.fa"
## 
## [[10]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_aegypti_Ago1_Exp6B_trim_clip.fa"
## 
## [[11]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_aegypti_Ago2_Exp6A_trim_clip.fa"
## 
## [[12]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_aegypti_Ago2_Exp6B_trim_clip.fa"
## 
## [[13]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_aegypti_mIgG_Exp6A_trim_clip.fa"
## 
## [[14]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_aegypti_mIgG_Exp6B_trim_clip.fa"
## 
## [[15]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_aegypti_rIgG_Exp6A_trim_clip.fa"
## 
## [[16]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_aegypti_rIgG_Exp6B_trim_clip.fa"

These reads are now ready to map to your genome

Build a Bowtie2 genome index and align to genome using Rbowtie2.

Build Bowtie2 index of reference genome, AaegL5 in this vignette.
Index documentation; see also CTK processing vignette.

Align reads to genome by bowtie.
format: format of input sequences, fasta (default) or fastq; threads: number of threads used, 1 (default); mode: “genome_map” (default, for mapping reads to a genome) or “reverse_map” (for mapping short sequences such as miRNAs to reads); mode can be set to NULL to set parameters manually
see bowtie_align documentation; see also Rbowtie2

#you will need to get the genome fasta of your organism and input the path to this fasta
genomeFasta <- paste0(baseDir, "/Aedes-aegypti-LVP_AGWG_CHROMOSOMES_AaegL5.fa")

#make index; this step may take a long time and is therefore commented out but shown to illustrate how to execute the function
#building the index only needs to be done the first time you map to a given genome

#genomeIndex <- bowtie2_index(genomeFasta)

#If your genome index is already made, you can simply point to the index
genomeIndex  <- paste0(baseDir, "/Aedes-aegypti-LVP_AGWG_CHROMOSOMES_AaegL5")

#Here I am selecting just a few samples for illustrative purposes. To run all your samples, you would do:
#tomap <- c(BrdU_split_rm5_rm3, samples_split_std_rm5_rm3)
tomap <- c(BrdU_split_rm5_rm3[1:2], samples_split_std_rm5_rm3[1:2])  # all I am doing here is taking samples 1-4 for each set of processed files (BrdU and standard) for mapping
tomap

## [[1]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_Aag2_Ago1_Exp6A_trim_clip.fa"
## 
## [[2]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_Aag2_Ago1_Exp6B_trim_clip.fa"
## 
## [[3]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001_collapse_Aag2_Ago1_Exp7A_rm5_clip.fa"
## 
## [[4]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001_collapse_Aag2_Ago1_Exp7B_rm5_clip.fa"

#map files to genome with default parameters: maxMismatches=1,seedSubString=18,threads=1,report_k=NULL
bams <- lapply(tomap, bowtie_align, genomeIndex, mode = "genome_map")
bams

## [[1]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_Aag2_Ago1_Exp6A_trim_clip.bam"
## 
## [[2]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_Aag2_Ago1_Exp6B_trim_clip.bam"
## 
## [[3]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001_collapse_Aag2_Ago1_Exp7A_rm5_clip.bam"
## 
## [[4]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001_collapse_Aag2_Ago1_Exp7B_rm5_clip.bam"

Convert to beds, find peaks, and build count matrix

Convert to beds

CLIPflexR documentation

beds <- lapply(bams, bamtobed)
beds

## [[1]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_Aag2_Ago1_Exp6A_trim_clip.bed"
## 
## [[2]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG091819_S1_L001_R1_001_collapse_rm5_Aag2_Ago1_Exp6B_trim_clip.bed"
## 
## [[3]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001_collapse_Aag2_Ago1_Exp7A_rm5_clip.bed"
## 
## [[4]]
## [1] "/Users/kathryn/Reprocess_all_paper_datasets/test_processing_vignette/KRG092419_S1_L001_R1_001_collapse_Aag2_Ago1_Exp7B_rm5_clip.bed"

Make tag directories and findpeaks (wrapping HOMER’s MakeTagDirectory and findpeaks)

Call significant peaks in each sample using R-wrapped homer_peaks from HOMER makeTagDirectory & HOMER findpeaks.
style: type of HOMER peak calling, “factor” (default); foldEnrichmentOverLocal: fold enrichment over local tag count, 2 (default); localSize:region to check for local tag enrichment, 10000 (default); strand: find peaks using tags on “seperate” (default); minDist: minimum distance between peaks, 50 (default); size: peak size, 10 (default); fragLength: approximate fragment length, 10 (default); genomeSize: NULL=2e9 (default, human or mouse), or set for your genome.
CLIPflexR documentation

#you may want to play with these parameters depending on your protein of interest, and ensure that coverage lines up with the peaks you have called
peaks <- lapply(beds, homer_peaks, fragLength=25)

Make count matrix of peaks, starting with reading in HOMER peak files and making a matrix that indicated which peaks were called in each sample

#read in peak files and convert to GRanges
read_peaks <- lapply(peaks,function(x)read.delim(x,sep="\t",comment.char = "#",h=FALSE))
read_peaksGR <- lapply(read_peaks,
                       function(x)GRanges(x$V2,IRanges(x$V3,x$V4),strand = x$V5))
names(read_peaksGR) <- gsub( paste0(baseDir,"/"), "",  (unlist(peaks)))
read_peaksGRList <- GRangesList(read_peaksGR)
GRSet <- unlist(read_peaksGRList)
nGRSet <- reduce(GRSet)
peakmat <- do.call(cbind,lapply(read_peaksGRList,
                                function(x)
                                  nGRSet %over% x))
mcols(nGRSet) <- peakmat

#now you have a peakmat where rows are a reduced set of peaks (without an identifier for the time being), and each column  is whether or not the peak was called in that sample
head(peakmat)

##      KRG091819_S1_L001_R1_001_collapse_rm5_Aag2_Ago1_Exp6A_trim_clip/peaks.txt
## [1,]                                                                     FALSE
## [2,]                                                                     FALSE
## [3,]                                                                     FALSE
## [4,]                                                                      TRUE
## [5,]                                                                     FALSE
## [6,]                                                                      TRUE
##      KRG091819_S1_L001_R1_001_collapse_rm5_Aag2_Ago1_Exp6B_trim_clip/peaks.txt
## [1,]                                                                     FALSE
## [2,]                                                                     FALSE
## [3,]                                                                     FALSE
## [4,]                                                                     FALSE
## [5,]                                                                      TRUE
## [6,]                                                                     FALSE
##      KRG092419_S1_L001_R1_001_collapse_Aag2_Ago1_Exp7A_rm5_clip/peaks.txt
## [1,]                                                                FALSE
## [2,]                                                                 TRUE
## [3,]                                                                 TRUE
## [4,]                                                                FALSE
## [5,]                                                                FALSE
## [6,]                                                                FALSE
##      KRG092419_S1_L001_R1_001_collapse_Aag2_Ago1_Exp7B_rm5_clip/peaks.txt
## [1,]                                                                 TRUE
## [2,]                                                                FALSE
## [3,]                                                                FALSE
## [4,]                                                                FALSE
## [5,]                                                                 TRUE
## [6,]                                                                FALSE

The next step is to countFromBed to append the number of reads at each peak location, by sample

CLIPflexR documentation

#defaults are: notStranded=TRUE,interFeature=FALSE
countmat <- lapply(beds,countFromBed,GR=nGRSet)

## Warning in .Seqinfo.mergexy(x, y): Each of the 2 combined objects has sequence levels not in the other:
##   - in 'x': NIGP01000917, NIGP01002025
##   - in 'y': NIGP01002089, NIGP01000727, NIGP01000475, NIGP01002221, NIGP01001492, NIGP01000892, NIGP01000201, NIGP01001727, NIGP01001982, NIGP01000101, NIGP01002078, NIGP01001086, NIGP01002082, NIGP01002045, NIGP01002203, NIGP01000451, NIGP01000495, NIGP01002020, NIGP01001844, NIGP01002286, NIGP01002127, NIGP01000184, NIGP01000944, NIGP01000304, NIGP01000130, NIGP01001935, NIGP01000647, NIGP01002035, NIGP01001914, NIGP01001774, NIGP01000058, NIGP01000662, NIGP01000976, NIGP01000253, NIGP01001770, NIGP01000416, NIGP01001671, NIGP01001712, NIGP01002113, NIGP01001514, NIGP01000379, NIGP01000036, NIGP01002034, NIGP01000552, NIGP01000094, NIGP01001837, NIGP01001551, NIGP01000525, NIGP01002215, NIGP01001678, NIGP01000638, NIGP01001599, NIGP01000106, NIGP01001294, NIGP01002178, NIGP01000256, NIGP01000283, NIGP01001031, NIGP01002155, NIGP01000077, NIGP01001793, NIGP01000371, NIGP01000658, NIGP01001480, NIGP01000848, NIGP01000415, NIGP01000180, NIGP01001039, NIGP01000110, NIGP01000242, NIGP01001503, NIGP01001976, NIGP01000537, NIGP01000565, NIGP01000807, NIGP01002168, NIGP01000482, NIGP01002289, NIGP01000751, NIGP01001945, NIGP01000574, NIGP01002209, NIGP01001759, NIGP01000402, NIGP01000198, NIGP01000292, NIGP01002029, NIGP01000294, NIGP01000909, NIGP01000139, NIGP01000282, NIGP01001786, NIGP01000376, NIGP01001698, NIGP01001206, NIGP01002240, NIGP01001030, NIGP01001414, NIGP01001750, NIGP01002188, NIGP01000072, NIGP01000080, NIGP01002269, NIGP01000033, NIGP01001661, NIGP01002234, NIGP01000761, NIGP01000063, NIGP01002251, NIGP01002176, NIGP01000433, NIGP01000034, NIGP01000159, NIGP01000575, NIGP01002300, NIGP01001458, NIGP01001507, NIGP01000182, NIGP01000941, NIGP01000188, NIGP01002297, NIGP01000697, NIGP01000629, NIGP01001062, NIGP01001508, NIGP01002230, NIGP01000197, NIGP01000266, NIGP01000699, NIGP01000541, NIGP01001755, NIGP01001696, NIGP01000656, NIGP01000563, NIGP01000888, NIGP01000327, NIGP01001988, NIGP01001641, NIGP01001846, NIGP01001908, NIGP01000986, NIGP01001769, NIGP01000440, NIGP01002046, NIGP01000505, NIGP01002152, NIGP01000627, NIGP01000598, NIGP01000274, NIGP01000414, NIGP01002253, NIGP01001029, NIGP01000599, NIGP01001510, NIGP01001887, NIGP01001615, NIGP01000650, NIGP01000735, NIGP01002199, NIGP01001036, NIGP01002153, NIGP01001949, NIGP01002196, NIGP01000407, NIGP01000437, NIGP01001919, NIGP01000809, NIGP01001929, NIGP01001703, NIGP01001866, NIGP01000504, NIGP01001327, NIGP01002058, NIGP01000779, NIGP01001557, NIGP01002024, NIGP01000859, NIGP01000206, NIGP01000189, NIGP01000210, NIGP01001019, NIGP01001717, NIGP01002087, NIGP01001650, NIGP01001845, NIGP01000361, NIGP01000579, NIGP01001741, NIGP01000205, NIGP01000802, NIGP01000548, NIGP01001757, NIGP01000356, NIGP01000893, NIGP01000637, NIGP01001469, NIGP01000780, NIGP01001915, NIGP01001943, NIGP01000335, NIGP01001933, NIGP01000920, NIGP01000863, NIGP01000417, NIGP01000845, NIGP01001008, NIGP01001803, NIGP01000039, NIGP01001816, NIGP01000665, NIGP01000818, NIGP01000521, NIGP01000578, NIGP01000532, NIGP01000821, NIGP01001546, NIGP01002242, NIGP01000384, NIGP01000295, NIGP01000408, NIGP01001940, NIGP01001566, NIGP01000865, NIGP01000646, NIGP01000187, NIGP01000962, NIGP01001024, NIGP01001076, NIGP01000497, NIGP01000916, NIGP01001927, NIGP01000791, NIGP01000760, NIGP01001028, NIGP01000124, NIGP01002055, NIGP01000212, NIGP01001898, NIGP01000149, NIGP01002044, NIGP01000363, NIGP01000904, NIGP01002133, NIGP01000123, NIGP01001567, NIGP01001956, NIGP01000232, NIGP01002275, NIGP01000142, NIGP01000413, NIGP01001736, NIGP01000728, NIGP01000608, NIGP01000290, NIGP01000477, NIGP01000806, NIGP01000315, NIGP01001858, NIGP01000577, NIGP01001066, NIGP01001934, NIGP01001767, NIGP01000573, NIGP01000648, NIGP01000747, NIGP01000397, NIGP01002103, NIGP01000600, NIGP01002121, NIGP01000254, NIGP01001977, NIGP01000959, NIGP01000009, NIGP01000640, NIGP01000061, NIGP01002278, NIGP01001893, NIGP01001584, NIGP01000218, NIGP01000148, NIGP01000711, NIGP01001875, NIGP01001085, NIGP01000463, NIGP01001536, NIGP01001672, NIGP01000450, NIGP01000736, NIGP01000281, NIGP01000031, NIGP01001758, NIGP01000840, NIGP01001961, NIGP01001925, NIGP01001554, NIGP01001764, NIGP01000098, NIGP01000457, NIGP01001690, NIGP01001534, NIGP01001942, NIGP01000216, NIGP01000965, NIGP01000328, NIGP01001909, NIGP01001392, NIGP01000522, NIGP01000213, NIGP01001593, NIGP01000464, NIGP01000456, NIGP01001540, NIGP01002260, NIGP01000279, NIGP01001061, NIGP01002191, NIGP01002064, NIGP01000486, NIGP01000775, NIGP01001847, NIGP01001688, NIGP01000963, NIGP01002181, NIGP01002014, NIGP01000424, NIGP01002179, NIGP01001077, NIGP01000870, NIGP01000580, NIGP01000056, NIGP01001498, NIGP01000639, NIGP01000768, NIGP01000745, NIGP01001781, NIGP01000813, NIGP01000666, NIGP01002099, NIGP01000092, NIGP01000885, NIGP01000700, NIGP01001634, NIGP01000381, NIGP01000645, NIGP01000958, NIGP01000620, NIGP01000263, NIGP01000896, NIGP01000046, NIGP01000025, NIGP01000378, NIGP01000403, NIGP01001639, NIGP01000185, NIGP01001561, NIGP01001601, NIGP01002009, NIGP01000095, NIGP01001704, NIGP01001053, NIGP01000194, NIGP01000866, NIGP01001497, NIGP01000568, NIGP01001017, NIGP01002126, NIGP01000369, NIGP01000020, NIGP01001573, NIGP01000291, NIGP01002170, NIGP01001430, NIGP01002096, NIGP01002167, NIGP01000257, NIGP01000996, NIGP01000670, NIGP01001810, NIGP01000476, NIGP01001928, NIGP01001082, NIGP01000950, NIGP01000759, NIGP01001635, NIGP01000737, NIGP01001415, NIGP01000060, NIGP01002207, NIGP01002077, NIGP01000680, NIGP01000052, NIGP01000121, NIGP01000113, NIGP01000144, NIGP01002154, NIGP01000975, NIGP01002157, NIGP01000269, NIGP01001072, NIGP01000088, NIGP01000583, NIGP01000426, NIGP01002163, NIGP01000581, NIGP01000125, NIGP01000247, NIGP01001791, NIGP01001499, NIGP01001676, NIGP01001711, NIGP01001902, NIGP01000480, NIGP01001035, NIGP01000709, NIGP01001763, NIGP01000367, NIGP01000597, NIGP01001068, NIGP01002146, NIGP01000473, NIGP01001748, NIGP01001728, NIGP01000823, NIGP01000103, NIGP01002106, NIGP01000014, NIGP01000134, NIGP01002065, NIGP01001707, NIGP01000203, NIGP01000657, NIGP01002263, NIGP01001737, NIGP01002169, NIGP01001819, NIGP01000280, NIGP01002151, NIGP01001833, NIGP01001708, NIGP01002273, NIGP01001084, NIGP01001059, NIGP01000596, NIGP01001807, NIGP01000375, NIGP01000507, NIGP01000714, NIGP01001673, NIGP01001880, NIGP01000852, NIGP01002042, NIGP01000489, NIGP01000757, NIGP01000340, NIGP01000447, NIGP01001805, NIGP01000374, NIGP01001700, NIGP01000713, NIGP01000069, NIGP01002122, NIGP01000734, NIGP01000047, NIGP01001958, NIGP01001890, NIGP01000453, NIGP01001994, NIGP01000805, NIGP01001749, NIGP01001979, NIGP01000621, NIGP01000694, NIGP01000972, NIGP01001968, NIGP01001839, NIGP01001989, NIGP01001960, NIGP01000209, NIGP01002187, NIGP01002001, NIGP01001813, NIGP01000150, NIGP01000991, NIGP01002084, NIGP01000514, NIGP01002039, NIGP01001739, NIGP01000157, NIGP01001842, NIGP01001073, NIGP01000005, NIGP01000285, NIGP01001281, NIGP01001572, NIGP01000786, NIGP01001867, NIGP01002158, NIGP01001521, NIGP01000084, NIGP01001517, NIGP01002189, NIGP01000919, NIGP01001530, NIGP01000635, NIGP01000107, NIGP01000204, NIGP01000644, NIGP01001014, NIGP01002228, NIGP01000228, NIGP01002264, NIGP01000557, NIGP01000329, NIGP01001630, NIGP01002110, NIGP01000246, NIGP01000049, NIGP01002128, NIGP01001439, NIGP01002101, NIGP01001952, NIGP01000999, NIGP01000411, NIGP01001146, NIGP01001074, NIGP01000750, NIGP01000096, NIGP01001811, NIGP01002308, NIGP01000718, NIGP01002193, NIGP01000938, NIGP01000053, NIGP01001041, NIGP01001595, NIGP01001752, NIGP01001733, NIGP01000547, NIGP01000710, NIGP01002177, NIGP01001230, NIGP01001533, NIGP01001691, NIGP01001301, NIGP01000667, NIGP01000617, NIGP01000441, NIGP01001240, NIGP01001991, NIGP01001046, NIGP01000041, NIGP01002130, NIGP01000633, NIGP01000907, NIGP01001018, NIGP01000832, NIGP01001519, NIGP01001153, NIGP01000017, NIGP01001217, NIGP01000654, NIGP01001795, NIGP01000422, NIGP01000967, NIGP01000942, NIGP01000609, NIGP01000560, NIGP01000401, NIGP01001081, NIGP01000652, NIGP01001783, NIGP01000177, NIGP01000882, NIGP01001067, NIGP01000326, NIGP01002229, NIGP01

## Warning in .Seqinfo.mergexy(x, y): Each of the 2 combined objects has sequence levels not in the other:
##   - in 'x': NIGP01001735
##   - in 'y': NIGP01002089, NIGP01000727, NIGP01000475, NIGP01002221, NIGP01001492, NIGP01000892, NIGP01000201, NIGP01001727, NIGP01001982, NIGP01000101, NIGP01002078, NIGP01001086, NIGP01002082, NIGP01002045, NIGP01002203, NIGP01000451, NIGP01000495, NIGP01002020, NIGP01001844, NIGP01002286, NIGP01002127, NIGP01000184, NIGP01000944, NIGP01000304, NIGP01000130, NIGP01001935, NIGP01000647, NIGP01002035, NIGP01001914, NIGP01001774, NIGP01000058, NIGP01000662, NIGP01000976, NIGP01000253, NIGP01001770, NIGP01000416, NIGP01001671, NIGP01001712, NIGP01002113, NIGP01001514, NIGP01000379, NIGP01000036, NIGP01002034, NIGP01000552, NIGP01000094, NIGP01001837, NIGP01001551, NIGP01000525, NIGP01002215, NIGP01001678, NIGP01000638, NIGP01001599, NIGP01000106, NIGP01001294, NIGP01002178, NIGP01000256, NIGP01000283, NIGP01001031, NIGP01002155, NIGP01000077, NIGP01001793, NIGP01000371, NIGP01000658, NIGP01001480, NIGP01000848, NIGP01000415, NIGP01001039, NIGP01000110, NIGP01000242, NIGP01001503, NIGP01001976, NIGP01000537, NIGP01000565, NIGP01000807, NIGP01002168, NIGP01000593, NIGP01000482, NIGP01002289, NIGP01000751, NIGP01001945, NIGP01000574, NIGP01002209, NIGP01001759, NIGP01000402, NIGP01000198, NIGP01000432, NIGP01000292, NIGP01002029, NIGP01000294, NIGP01000909, NIGP01000139, NIGP01000282, NIGP01001786, NIGP01000376, NIGP01001698, NIGP01001206, NIGP01002240, NIGP01001030, NIGP01001414, NIGP01001750, NIGP01002188, NIGP01000072, NIGP01000080, NIGP01002269, NIGP01000033, NIGP01001661, NIGP01002234, NIGP01000602, NIGP01000761, NIGP01000063, NIGP01002251, NIGP01002176, NIGP01000433, NIGP01000034, NIGP01000159, NIGP01000575, NIGP01002300, NIGP01001458, NIGP01001507, NIGP01000941, NIGP01000188, NIGP01002297, NIGP01000629, NIGP01001062, NIGP01001508, NIGP01002230, NIGP01000197, NIGP01000699, NIGP01000541, NIGP01001755, NIGP01000656, NIGP01000563, NIGP01000888, NIGP01000327, NIGP01001988, NIGP01001641, NIGP01001846, NIGP01001908, NIGP01000986, NIGP01001769, NIGP01000440, NIGP01002046, NIGP01002152, NIGP01000627, NIGP01000274, NIGP01000414, NIGP01002253, NIGP01001029, NIGP01000599, NIGP01001510, NIGP01001887, NIGP01001615, NIGP01000650, NIGP01000735, NIGP01002199, NIGP01001036, NIGP01002153, NIGP01001949, NIGP01002196, NIGP01000407, NIGP01000437, NIGP01001919, NIGP01000809, NIGP01001929, NIGP01001703, NIGP01001866, NIGP01000504, NIGP01001327, NIGP01002058, NIGP01000779, NIGP01001557, NIGP01000859, NIGP01000206, NIGP01000189, NIGP01000210, NIGP01001019, NIGP01001717, NIGP01002087, NIGP01001650, NIGP01001845, NIGP01000361, NIGP01000579, NIGP01001741, NIGP01000205, NIGP01000802, NIGP01000548, NIGP01000356, NIGP01000893, NIGP01001469, NIGP01000780, NIGP01001915, NIGP01001943, NIGP01000335, NIGP01001933, NIGP01000920, NIGP01000863, NIGP01000417, NIGP01000845, NIGP01001008, NIGP01001803, NIGP01000039, NIGP01001816, NIGP01000665, NIGP01000818, NIGP01000521, NIGP01000578, NIGP01000532, NIGP01000821, NIGP01001546, NIGP01002242, NIGP01000384, NIGP01000295, NIGP01000471, NIGP01000408, NIGP01001940, NIGP01001566, NIGP01000865, NIGP01000646, NIGP01000187, NIGP01000316, NIGP01001024, NIGP01001076, NIGP01000497, NIGP01000916, NIGP01000791, NIGP01000760, NIGP01001028, NIGP01000124, NIGP01001612, NIGP01002055, NIGP01000212, NIGP01001898, NIGP01000149, NIGP01002044, NIGP01000904, NIGP01000123, NIGP01001697, NIGP01001567, NIGP01001956, NIGP01000232, NIGP01002275, NIGP01000142, NIGP01000413, NIGP01001736, NIGP01000728, NIGP01000608, NIGP01000290, NIGP01000477, NIGP01000806, NIGP01000315, NIGP01001858, NIGP01000577, NIGP01001066, NIGP01001934, NIGP01001767, NIGP01000573, NIGP01000648, NIGP01000747, NIGP01000397, NIGP01002103, NIGP01000600, NIGP01002121, NIGP01000254, NIGP01001977, NIGP01000959, NIGP01000009, NIGP01000640, NIGP01001060, NIGP01002278, NIGP01001893, NIGP01000218, NIGP01000148, NIGP01000711, NIGP01001875, NIGP01001085, NIGP01001536, NIGP01001672, NIGP01000736, NIGP01000281, NIGP01000031, NIGP01001758, NIGP01000840, NIGP01001516, NIGP01001961, NIGP01001925, NIGP01001554, NIGP01001606, NIGP01000673, NIGP01001764, NIGP01002054, NIGP01000098, NIGP01000457, NIGP01002095, NIGP01001690, NIGP01001534, NIGP01001942, NIGP01000216, NIGP01000328, NIGP01001909, NIGP01001392, NIGP01001058, NIGP01000512, NIGP01000522, NIGP01000213, NIGP01000464, NIGP01000456, NIGP01001540, NIGP01002260, NIGP01001061, NIGP01002191, NIGP01002064, NIGP01000486, NIGP01000775, NIGP01001847, NIGP01001688, NIGP01001950, NIGP01000963, NIGP01002181, NIGP01002014, NIGP01000424, NIGP01002179, NIGP01001077, NIGP01000870, NIGP01000056, NIGP01001498, NIGP01000639, NIGP01000768, NIGP01000745, NIGP01001781, NIGP01000813, NIGP01000666, NIGP01000087, NIGP01002099, NIGP01000092, NIGP01000885, NIGP01000700, NIGP01001634, NIGP01000381, NIGP01000645, NIGP01000958, NIGP01000620, NIGP01000263, NIGP01000896, NIGP01000025, NIGP01000378, NIGP01000403, NIGP01001639, NIGP01000185, NIGP01001561, NIGP01001601, NIGP01002009, NIGP01000095, NIGP01001704, NIGP01000217, NIGP01001053, NIGP01000194, NIGP01000866, NIGP01001497, NIGP01002126, NIGP01000020, NIGP01001573, NIGP01000291, NIGP01002170, NIGP01001114, NIGP01000289, NIGP01001683, NIGP01002096, NIGP01002167, NIGP01000257, NIGP01000670, NIGP01001810, NIGP01000636, NIGP01000476, NIGP01001928, NIGP01001082, NIGP01000950, NIGP01000759, NIGP01001635, NIGP01000737, NIGP01001415, NIGP01000060, NIGP01002207, NIGP01002077, NIGP01000386, NIGP01000052, NIGP01000121, NIGP01000113, NIGP01000144, NIGP01002154, NIGP01000190, NIGP01002157, NIGP01000269, NIGP01001072, NIGP01000088, NIGP01000583, NIGP01000426, NIGP01002163, NIGP01000581, NIGP01000125, NIGP01000247, NIGP01001791, NIGP01001499, NIGP01001676, NIGP01001711, NIGP01001902, NIGP01000523, NIGP01001870, NIGP01001035, NIGP01000709, NIGP01001763, NIGP01000367, NIGP01000597, NIGP01001068, NIGP01002146, NIGP01000473, NIGP01001748, NIGP01001728, NIGP01000823, NIGP01000103, NIGP01002106, NIGP01000014, NIGP01000134, NIGP01002065, NIGP01000203, NIGP01002263, NIGP01001737, NIGP01001819, NIGP01002151, NIGP01001833, NIGP01000969, NIGP01002273, NIGP01001059, NIGP01002283, NIGP01001807, NIGP01000375, NIGP01000507, NIGP01000714, NIGP01001880, NIGP01000852, NIGP01000348, NIGP01002042, NIGP01000344, NIGP01000489, NIGP01000757, NIGP01000340, NIGP01000447, NIGP01001805, NIGP01000374, NIGP01000713, NIGP01000069, NIGP01002122, NIGP01000734, NIGP01000047, NIGP01001958, NIGP01001890, NIGP01000453, NIGP01001994, NIGP01000805, NIGP01001749, NIGP01002180, NIGP01001979, NIGP01000621, NIGP01000694, NIGP01000972, NIGP01001968, NIGP01001839, NIGP01000449, NIGP01001989, NIGP01001960, NIGP01000209, NIGP01000156, NIGP01002001, NIGP01000150, NIGP01000991, NIGP01000514, NIGP01000871, NIGP01001440, NIGP01000157, NIGP01001842, NIGP01000285, NIGP01001281, NIGP01001572, NIGP01000786, NIGP01001867, NIGP01002158, NIGP01001521, NIGP01000084, NIGP01002086, NIGP01001517, NIGP01002189, NIGP01000919, NIGP01001530, NIGP01000635, NIGP01000107, NIGP01000204, NIGP01001785, NIGP01000644, NIGP01001014, NIGP01000228, NIGP01002264, NIGP01000557, NIGP01000329, NIGP01000539, NIGP01001630, NIGP01001049, NIGP01002110, NIGP01002128, NIGP01001439, NIGP01002101, NIGP01002129, NIGP01001952, NIGP01000999, NIGP01000411, NIGP01000750, NIGP01000096, NIGP01001811, NIGP01002308, NIGP01000718, NIGP01002193, NIGP01001047, NIGP01000938, NIGP01000053, NIGP01001041, NIGP01001595, NIGP01001752, NIGP01000547, NIGP01000710, NIGP01002288, NIGP01002177, NIGP01001533, NIGP01001838, NIGP01002186, NIGP01001691, NIGP01001301, NIGP01000667, NIGP01000617, NIGP01000441, NIGP01001240, NIGP01001991, NIGP01001046, NIGP01000041, NIGP01002130, NIGP01000633, NIGP01000907, NIGP01001018, NIGP01001519, NIGP01000017, NIGP01001217, NIGP01000654, NIGP01001795, NIGP01000422, NIGP01000967, NIGP01000942, NIGP01000609, NIGP01000428, NIGP01000560, NIGP01000401, NIGP01001081, NIGP01000177, NIGP01000882, NIGP01001067, NIGP01000326, NIGP01000843, NIGP01002118, NIGP01000663, NIGP01000310, NIGP01000233, NIGP01001828, NIGP01000465, NIGP01000953, NIGP01000649, NIGP01002298, NIGP01000394, NIGP01001743, NIGP01001558, NIGP01000377, NIGP01000970, NIGP01

## Warning in .Seqinfo.mergexy(x, y): Each of the 2 combined objects has sequence levels not in the other:
##   - in 'x': NIGP01000990
##   - in 'y': NIGP01002089, NIGP01000727, NIGP01000475, NIGP01002221, NIGP01001492, NIGP01000892, NIGP01000201, NIGP01001727, NIGP01001982, NIGP01000101, NIGP01002078, NIGP01001086, NIGP01002082, NIGP01002045, NIGP01002203, NIGP01000451, NIGP01000495, NIGP01002020, NIGP01001844, NIGP01002286, NIGP01002127, NIGP01000184, NIGP01000944, NIGP01000304, NIGP01000130, NIGP01001935, NIGP01000647, NIGP01002035, NIGP01001914, NIGP01001774, NIGP01000058, NIGP01000662, NIGP01000976, NIGP01000253, NIGP01001770, NIGP01000416, NIGP01001671, NIGP01001712, NIGP01002113, NIGP01001514, NIGP01000379, NIGP01000036, NIGP01002034, NIGP01000552, NIGP01001837, NIGP01001551, NIGP01000525, NIGP01002215, NIGP01001678, NIGP01000638, NIGP01001599, NIGP01000106, NIGP01001294, NIGP01002178, NIGP01000256, NIGP01000283, NIGP01001031, NIGP01002155, NIGP01000077, NIGP01001793, NIGP01000371, NIGP01000658, NIGP01001480, NIGP01000848, NIGP01000415, NIGP01000180, NIGP01001039, NIGP01000110, NIGP01000242, NIGP01001503, NIGP01001976, NIGP01000537, NIGP01000565, NIGP01000807, NIGP01002168, NIGP01000593, NIGP01000482, NIGP01002289, NIGP01000751, NIGP01001945, NIGP01000574, NIGP01002209, NIGP01001759, NIGP01000402, NIGP01000198, NIGP01000432, NIGP01002029, NIGP01000294, NIGP01000909, NIGP01000139, NIGP01000282, NIGP01001786, NIGP01000376, NIGP01001698, NIGP01001206, NIGP01002240, NIGP01001030, NIGP01001414, NIGP01001750, NIGP01002188, NIGP01000072, NIGP01000080, NIGP01002269, NIGP01000033, NIGP01001661, NIGP01002234, NIGP01000602, NIGP01000761, NIGP01000063, NIGP01002251, NIGP01002176, NIGP01000433, NIGP01000034, NIGP01000159, NIGP01002300, NIGP01001458, NIGP01001507, NIGP01000182, NIGP01000941, NIGP01000188, NIGP01002297, NIGP01000697, NIGP01000629, NIGP01001062, NIGP01001508, NIGP01002230, NIGP01000197, NIGP01000266, NIGP01000699, NIGP01000541, NIGP01001755, NIGP01001696, NIGP01000656, NIGP01000563, NIGP01000888, NIGP01000327, NIGP01001988, NIGP01001641, NIGP01001846, NIGP01001908, NIGP01000986, NIGP01001769, NIGP01000440, NIGP01002046, NIGP01000505, NIGP01002152, NIGP01000627, NIGP01000598, NIGP01000274, NIGP01000414, NIGP01002253, NIGP01001029, NIGP01000599, NIGP01001510, NIGP01001887, NIGP01000650, NIGP01000735, NIGP01002199, NIGP01001036, NIGP01002153, NIGP01001949, NIGP01002196, NIGP01000407, NIGP01001919, NIGP01000809, NIGP01001929, NIGP01001703, NIGP01001866, NIGP01002058, NIGP01000779, NIGP01001557, NIGP01002024, NIGP01000859, NIGP01000206, NIGP01000189, NIGP01000210, NIGP01001019, NIGP01001717, NIGP01002087, NIGP01001650, NIGP01001965, NIGP01001845, NIGP01000361, NIGP01000579, NIGP01001741, NIGP01000205, NIGP01000802, NIGP01001757, NIGP01000356, NIGP01000893, NIGP01000637, NIGP01001469, NIGP01000780, NIGP01001915, NIGP01001943, NIGP01000335, NIGP01001933, NIGP01000920, NIGP01000863, NIGP01000417, NIGP01000845, NIGP01001008, NIGP01000039, NIGP01001816, NIGP01000665, NIGP01000818, NIGP01000521, NIGP01000578, NIGP01000532, NIGP01000821, NIGP01001546, NIGP01002242, NIGP01000384, NIGP01000295, NIGP01000408, NIGP01001940, NIGP01001566, NIGP01000865, NIGP01000187, NIGP01000962, NIGP01000316, NIGP01001024, NIGP01001076, NIGP01000497, NIGP01000916, NIGP01001927, NIGP01000791, NIGP01000124, NIGP01001612, NIGP01002055, NIGP01000212, NIGP01001898, NIGP01002044, NIGP01000363, NIGP01000904, NIGP01002133, NIGP01000123, NIGP01001697, NIGP01001567, NIGP01001956, NIGP01002275, NIGP01000142, NIGP01000413, NIGP01001736, NIGP01000728, NIGP01000290, NIGP01000477, NIGP01000806, NIGP01000315, NIGP01001858, NIGP01000577, NIGP01001066, NIGP01001934, NIGP01001767, NIGP01000648, NIGP01000747, NIGP01000397, NIGP01002103, NIGP01000600, NIGP01002121, NIGP01000254, NIGP01001977, NIGP01000959, NIGP01000009, NIGP01000640, NIGP01000061, NIGP01001893, NIGP01000218, NIGP01000711, NIGP01001875, NIGP01001085, NIGP01000463, NIGP01001672, NIGP01000450, NIGP01000281, NIGP01000031, NIGP01001758, NIGP01000840, NIGP01001961, NIGP01001925, NIGP01001554, NIGP01001606, NIGP01001764, NIGP01002054, NIGP01000098, NIGP01000457, NIGP01002095, NIGP01001690, NIGP01001534, NIGP01001942, NIGP01000216, NIGP01000965, NIGP01000328, NIGP01001909, NIGP01001392, NIGP01001058, NIGP01000512, NIGP01000213, NIGP01001593, NIGP01000464, NIGP01000456, NIGP01001540, NIGP01002260, NIGP01000279, NIGP01001061, NIGP01002064, NIGP01000775, NIGP01001847, NIGP01001950, NIGP01000963, NIGP01002181, NIGP01000424, NIGP01002179, NIGP01001077, NIGP01000870, NIGP01000580, NIGP01000056, NIGP01001498, NIGP01000639, NIGP01000768, NIGP01000745, NIGP01001781, NIGP01000666, NIGP01000087, NIGP01002099, NIGP01000092, NIGP01000885, NIGP01000700, NIGP01001634, NIGP01000381, NIGP01000645, NIGP01000958, NIGP01000263, NIGP01000896, NIGP01000046, NIGP01000378, NIGP01000403, NIGP01001639, NIGP01001561, NIGP01002009, NIGP01000095, NIGP01001704, NIGP01000217, NIGP01001053, NIGP01000194, NIGP01000866, NIGP01001497, NIGP01002126, NIGP01001573, NIGP01001114, NIGP01000289, NIGP01002096, NIGP01002167, NIGP01000257, NIGP01000996, NIGP01000670, NIGP01001810, NIGP01000476, NIGP01001928, NIGP01001082, NIGP01000950, NIGP01000759, NIGP01001635, NIGP01000737, NIGP01001415, NIGP01000060, NIGP01002207, NIGP01002077, NIGP01000386, NIGP01000052, NIGP01000121, NIGP01000113, NIGP01000144, NIGP01002157, NIGP01000269, NIGP01000088, NIGP01000583, NIGP01000426, NIGP01000167, NIGP01002163, NIGP01000581, NIGP01000125, NIGP01000247, NIGP01001791, NIGP01001499, NIGP01001676, NIGP01001711, NIGP01001902, NIGP01000523, NIGP01000480, NIGP01001870, NIGP01000709, NIGP01000367, NIGP01001068, NIGP01002146, NIGP01000473, NIGP01001748, NIGP01001728, NIGP01000823, NIGP01002106, NIGP01000014, NIGP01000134, NIGP01002065, NIGP01000203, NIGP01000657, NIGP01002263, NIGP01001737, NIGP01001819, NIGP01002151, NIGP01001833, NIGP01000969, NIGP01001059, NIGP01000596, NIGP01001807, NIGP01000375, NIGP01000507, NIGP01000714, NIGP01001880, NIGP01002042, NIGP01000344, NIGP01000489, NIGP01000757, NIGP01000447, NIGP01001805, NIGP01000374, NIGP01000713, NIGP01002122, NIGP01000734, NIGP01000047, NIGP01001958, NIGP01000453, NIGP01000805, NIGP01001749, NIGP01002180, NIGP01001979, NIGP01000621, NIGP01000694, NIGP01000972, NIGP01001968, NIGP01001839, NIGP01001989, NIGP01001960, NIGP01000209, NIGP01002187, NIGP01000156, NIGP01002001, NIGP01001813, NIGP01000150, NIGP01000991, NIGP01002084, NIGP01000514, NIGP01002039, NIGP01000871, NIGP01001440, NIGP01001739, NIGP01001713, NIGP01000157, NIGP01001842, NIGP01001073, NIGP01000005, NIGP01000285, NIGP01001572, NIGP01000786, NIGP01001867, NIGP01001521, NIGP01000084, NIGP01002086, NIGP01001338, NIGP01001517, NIGP01000919, NIGP01000635, NIGP01000107, NIGP01001785, NIGP01001014, NIGP01002228, NIGP01000228, NIGP01002264, NIGP01000329, NIGP01000539, NIGP01001630, NIGP01001049, NIGP01002110, NIGP01002128, NIGP01001439, NIGP01002101, NIGP01002129, NIGP01001952, NIGP01000411, NIGP01001146, NIGP01001074, NIGP01000750, NIGP01000096, NIGP01001811, NIGP01000718, NIGP01002193, NIGP01000938, NIGP01000053, NIGP01001041, NIGP01001595, NIGP01001752, NIGP01000547, NIGP01000710, NIGP01002288, NIGP01002177, NIGP01001533, NIGP01001838, NIGP01001691, NIGP01001301, NIGP01000667, NIGP01000617, NIGP01000441, NIGP01001240, NIGP01001991, NIGP01001046, NIGP01000041, NIGP01002130, NIGP01000633, NIGP01000907, NIGP01001018, NIGP01001519, NIGP01001821, NIGP01001153, NIGP01001217, NIGP01000654, NIGP01001795, NIGP01000422, NIGP01000967, NIGP01000942, NIGP01000609, NIGP01000560, NIGP01000401, NIGP01001081, NIGP01000652, NIGP01000055, NIGP01000177, NIGP01000882, NIGP01001067, NIGP01000326, NIGP01002229, NIGP01000616, NIGP01000238, NIGP01000742, NIGP01000843, NIGP01002118, NIGP01001178, NIGP01000310, NIGP01000979, NIGP01000233, NIGP01000465, NIGP01000953, NIGP01000649, NIGP01001993, NIGP01002298, NIGP01001743, NIGP01001558, NIGP01000377, NIGP01000970, NIGP01000948, NIGP01001216, NIGP01000436, NIGP01000715, NIGP01000261, NIGP01001901, NIGP01000481, NIGP01000674, NIGP01001777, NIGP01000877, NIGP01000632, NIGP01000546, NIGP01000752, NIGP01000010, NIGP01000421, NIGP01000501, NIGP01000082, NIGP01000089, NIGP01001897, NIGP01001710, NIGP01

countmat <- do.call(cbind,countmat)
colnames(countmat) <- basename(unlist(beds))

#now bind nGRSet, a GRanges with peak ranges and TRUE/FALSE at each peak, with countmat, the number of reads at each peak
mcols(nGRSet) <- cbind(as.data.frame(mcols(nGRSet)),countmat)

#you can convert this GRanges object to a dataframe
peaksDF <- as.data.frame(nGRSet)
peaksDF$peakID <- paste0(peaksDF$seqnames, ":", peaksDF$start, "_", peaksDF$end, ":", peaksDF$strand)
head(peaksDF)

##   seqnames   start     end width strand
## 1        1  301043  301053    11      +
## 2        1  580903  580913    11      +
## 3        1 1183109 1183119    11      +
## 4        1 1386212 1386222    11      +
## 5        1 1947557 1947569    13      +
## 6        1 1947625 1947635    11      +
##   KRG091819_S1_L001_R1_001_collapse_rm5_Aag2_Ago1_Exp6A_trim_clip.peaks.txt
## 1                                                                     FALSE
## 2                                                                     FALSE
## 3                                                                     FALSE
## 4                                                                      TRUE
## 5                                                                     FALSE
## 6                                                                      TRUE
##   KRG091819_S1_L001_R1_001_collapse_rm5_Aag2_Ago1_Exp6B_trim_clip.peaks.txt
## 1                                                                     FALSE
## 2                                                                     FALSE
## 3                                                                     FALSE
## 4                                                                     FALSE
## 5                                                                      TRUE
## 6                                                                     FALSE
##   KRG092419_S1_L001_R1_001_collapse_Aag2_Ago1_Exp7A_rm5_clip.peaks.txt
## 1                                                                FALSE
## 2                                                                 TRUE
## 3                                                                 TRUE
## 4                                                                FALSE
## 5                                                                FALSE
## 6                                                                FALSE
##   KRG092419_S1_L001_R1_001_collapse_Aag2_Ago1_Exp7B_rm5_clip.peaks.txt
## 1                                                                 TRUE
## 2                                                                FALSE
## 3                                                                FALSE
## 4                                                                FALSE
## 5                                                                 TRUE
## 6                                                                FALSE
##   KRG091819_S1_L001_R1_001_collapse_rm5_Aag2_Ago1_Exp6A_trim_clip.bed
## 1                                                                   0
## 2                                                                   0
## 3                                                                   0
## 4                                                                   3
## 5                                                                   3
## 6                                                                   3
##   KRG091819_S1_L001_R1_001_collapse_rm5_Aag2_Ago1_Exp6B_trim_clip.bed
## 1                                                                   0
## 2                                                                   0
## 3                                                                   0
## 4                                                                   1
## 5                                                                   4
## 6                                                                   1
##   KRG092419_S1_L001_R1_001_collapse_Aag2_Ago1_Exp7A_rm5_clip.bed
## 1                                                              0
## 2                                                              3
## 3                                                              3
## 4                                                              0
## 5                                                              5
## 6                                                              2
##   KRG092419_S1_L001_R1_001_collapse_Aag2_Ago1_Exp7B_rm5_clip.bed
## 1                                                              3
## 2                                                              0
## 3                                                              3
## 4                                                              0
## 5                                                              5
## 6                                                              0
##                peakID
## 1   1:301043_301053:+
## 2   1:580903_580913:+
## 3 1:1183109_1183119:+
## 4 1:1386212_1386222:+
## 5 1:1947557_1947569:+
## 6 1:1947625_1947635:+

now you have your count matrix!

you can perform operations on sample groups to filter, visualize data, annotate your peaks, and more…

For example, if you are dealing with a custom genome without pre-built genomes and annotation, you can still easily annotate your peaks
Here we provide an example using ChIPseeker, which you can also use to visualize genomic annotation (see ChIPseeker vignette for alternate visualizations)

#load required packages
library(ChIPseeker)
library(GenomicFeatures)

gtffile <- "/Users/kathryn/Aedes-aegypti-LVP_AGWG_BASEFEATURES_AaegL5.2.gtf"  #custom gtf or gff
 
TxDb <- makeTxDbFromGFF(gtffile)

## Import genomic features from the file as a GRanges object ... OK
## Prepare the 'metadata' data frame ... OK
## Make the TxDb object ...

## Warning in .get_cds_IDX(type, phase): The "phase" metadata column contains non-NA values for features of
##   type stop_codon. This information was ignored.

## OK

allanno <- annotatePeak(nGRSet,
                              TxDb=TxDb,
                              sameStrand=FALSE,
                              genomicAnnotationPriority = c("3UTR", "5UTR", "Exon",
                                                            "Intron", "Promoter", "Downstream", "Intergenic"),
                              overlap = "all")

## >> preparing features information...      2021-03-16 02:33:31 
## >> identifying nearest features...        2021-03-16 02:33:31

## Warning in .Seqinfo.mergexy(x, y): Each of the 2 combined objects has sequence levels not in the other:
##   - in 'x': NIGP01000081, NIGP01000723, NIGP01001312, NIGP01001552, NIGP01001579, NIGP01001581, NIGP01001642, NIGP01001658, NIGP01001735, NIGP01001852, NIGP01002067, NIGP01000332, NIGP01000588, NIGP01001651, NIGP01001665, NIGP01001990, NIGP01000226, NIGP01000358, NIGP01000825, NIGP01000917, NIGP01000946, NIGP01001002, NIGP01001056, NIGP01001101, NIGP01001349, NIGP01001814, NIGP01002025, NIGP01001900
##   - in 'y': NIGP01000006, NIGP01000009, NIGP01000010, NIGP01000015, NIGP01000025, NIGP01000028, NIGP01000033, NIGP01000036, NIGP01000043, NIGP01000049, NIGP01000051, NIGP01000052, NIGP01000055, NIGP01000056, NIGP01000062, NIGP01000063, NIGP01000064, NIGP01000073, NIGP01000075, NIGP01000076, NIGP01000077, NIGP01000079, NIGP01000080, NIGP01000083, NIGP01000087, NIGP01000094, NIGP01000096, NIGP01000101, NIGP01000102, NIGP01000104, NIGP01000106, NIGP01000107, NIGP01000110, NIGP01000115, NIGP01000116, NIGP01000117, NIGP01000121, NIGP01000123, NIGP01000124, NIGP01000125, NIGP01000129, NIGP01000130, NIGP01000133, NIGP01000134, NIGP01000137, NIGP01000139, NIGP01000144, NIGP01000149, NIGP01000156, NIGP01000158, NIGP01000159, NIGP01000172, NIGP01000177, NIGP01000180, NIGP01000183, NIGP01000184, NIGP01000185, NIGP01000186, NIGP01000187, NIGP01000188, NIGP01000189, NIGP01000197, NIGP01000198, NIGP01000200, NIGP01000201, NIGP01000203, NIGP01000204, NIGP01000205, NIGP01000206, NIGP01000207, NIGP01000210, NIGP01000212, NIGP01000213, NIGP01000216, NIGP01000218, NIGP01000220, NIGP01000221, NIGP01000222, NIGP01000223, NIGP01000225, NIGP01000228, NIGP01000230, NIGP01000236, NIGP01000238, NIGP01000239, NIGP01000240, NIGP01000244, NIGP01000247, NIGP01000248, NIGP01000250, NIGP01000253, NIGP01000254, NIGP01000256, NIGP01000257, NIGP01000262, NIGP01000263, NIGP01000265, NIGP01000267, NIGP01000270, NIGP01000280, NIGP01000281, NIGP01000288, NIGP01000290, NIGP01000292, NIGP01000294, NIGP01000295, NIGP01000297, NIGP01000298, NIGP01000309, NIGP01000310, NIGP01000312, NIGP01000315, NIGP01000319, NIGP01000324, NIGP01000325, NIGP01000329, NIGP01000330, NIGP01000345, NIGP01000349, NIGP01000351, NIGP01000352, NIGP01000356, NIGP01000357, NIGP01000359, NIGP01000363, NIGP01000365, NIGP01000371, NIGP01000374, NIGP01000375, NIGP01000376, NIGP01000377, NIGP01000379, NIGP01000383, NIGP01000386, NIGP01000396, NIGP01000397, NIGP01000407, NIGP01000410, NIGP01000412, NIGP01000413, NIGP01000415, NIGP01000417, NIGP01000421, NIGP01000422, NIGP01000424, NIGP01000428, NIGP01000430, NIGP01000432, NIGP01000434, NIGP01000437, NIGP01000440, NIGP01000441, NIGP01000445, NIGP01000451, NIGP01000452, NIGP01000461, NIGP01000463, NIGP01000465, NIGP01000466, NIGP01000475, NIGP01000481, NIGP01000482, NIGP01000489, NIGP01000490, NIGP01000495, NIGP01000497, NIGP01000501, NIGP01000507, NIGP01000514, NIGP01000515, NIGP01000518, NIGP01000519, NIGP01000520, NIGP01000521, NIGP01000524, NIGP01000525, NIGP01000530, NIGP01000531, NIGP01000532, NIGP01000535, NIGP01000536, NIGP01000537, NIGP01000539, NIGP01000541, NIGP01000552, NIGP01000553, NIGP01000556, NIGP01000557, NIGP01000560, NIGP01000561, NIGP01000566, NIGP01000569, NIGP01000570, NIGP01000571, NIGP01000573, NIGP01000574, NIGP01000579, NIGP01000590, NIGP01000591, NIGP01000596, NIGP01000598, NIGP01000599, NIGP01000600, NIGP01000601, NIGP01000602, NIGP01000604, NIGP01000608, NIGP01000610, NIGP01000612, NIGP01000622, NIGP01000627, NIGP01000630, NIGP01000633, NIGP01000634, NIGP01000635, NIGP01000639, NIGP01000640, NIGP01000648, NIGP01000649, NIGP01000650, NIGP01000651, NIGP01000657, NIGP01000658, NIGP01000660, NIGP01000662, NIGP01000665, NIGP01000666, NIGP01000667, NIGP01000669, NIGP01000673, NIGP01000676, NIGP01000681, NIGP01000684, NIGP01000686, NIGP01000687, NIGP01000690, NIGP01000692, NIGP01000695, NIGP01000699, NIGP01000702, NIGP01000703, NIGP01000706, NIGP01000709, NIGP01000710, NIGP01000712, NIGP01000714, NIGP01000715, NIGP01000716, NIGP01000718, NIGP01000721, NIGP01000724, NIGP01000727, NIGP01000730, NIGP01000734, NIGP01000737, NIGP01000740, NIGP01000745, NIGP01000751, NIGP01000754, NIGP01000756, NIGP01000760, NIGP01000761, NIGP01000775, NIGP01000778, NIGP01000779, NIGP01000781, NIGP01000782, NIGP01000783, NIGP01000791, NIGP01000799, NIGP01000802, NIGP01000806, NIGP01000807, NIGP01000811, NIGP01000812, NIGP01000813, NIGP01000818, NIGP01000821, NIGP01000822, NIGP01000823, NIGP01000826, NIGP01000827, NIGP01000835, NIGP01000843, NIGP01000846, NIGP01000847, NIGP01000848, NIGP01000849, NIGP01000850, NIGP01000856, NIGP01000859, NIGP01000860, NIGP01000861, NIGP01000867, NIGP01000870, NIGP01000882, NIGP01000883, NIGP01000885, NIGP01000892, NIGP01000896, NIGP01000900, NIGP01000904, NIGP01000905, NIGP01000906, NIGP01000910, NIGP01000915, NIGP01000920, NIGP01000921, NIGP01000923, NIGP01000929, NIGP01000932, NIGP01000937, NIGP01000938, NIGP01000939, NIGP01000941, NIGP01000942, NIGP01000944, NIGP01000947, NIGP01000948, NIGP01000952, NIGP01000954, NIGP01000957, NIGP01000958, NIGP01000959, NIGP01000960, NIGP01000976, NIGP01000977, NIGP01000978, NIGP01000980, NIGP01000983, NIGP01000986, NIGP01000991, NIGP01000996, NIGP01001003, NIGP01001006, NIGP01001008, NIGP01001010, NIGP01001013, NIGP01001015, NIGP01001018, NIGP01001022, NIGP01001023, NIGP01001024, NIGP01001031, NIGP01001032, NIGP01001035, NIGP01001039, NIGP01001041, NIGP01001043, NIGP01001046, NIGP01001047, NIGP01001053, NIGP01001055, NIGP01001057, NIGP01001059, NIGP01001061, NIGP01001068, NIGP01001070, NIGP01001072, NIGP01001074, NIGP01001075, NIGP01001081, NIGP01001082, NIGP01001083, NIGP01001085, NIGP01001086, NIGP01001092, NIGP01001097, NIGP01001100, NIGP01001102, NIGP01001108, NIGP01001110, NIGP01001119, NIGP01001126, NIGP01001136, NIGP01001165, NIGP01001173, NIGP01001180, NIGP01001185, NIGP01001186, NIGP01001196, NIGP01001197, NIGP01001208, NIGP01001211, NIGP01001214, NIGP01001216, NIGP01001230, NIGP01001233, NIGP01001237, NIGP01001238, NIGP01001240, NIGP01001243, NIGP01001247, NIGP01001249, NIGP01001254, NIGP01001255, NIGP01001256, NIGP01001262, NIGP01001263, NIGP01001282, NIGP01001285, NIGP01001287, NIGP01001295, NIGP01001296, NIGP01001297, NIGP01001301, NIGP01001311, NIGP01001322, NIGP01001325, NIGP01001329, NIGP01001335, NIGP01001341, NIGP01001343, NIGP01001348, NIGP01001350, NIGP01001352, NIGP01001353, NIGP01001355, NIGP01001363, NIGP01001365, NIGP01001372, NIGP01001373, NIGP01001374, NIGP01001377, NIGP01001382, NIGP01001386, NIGP01001397, NIGP01001398, NIGP01001407, NIGP01001408, NIGP01001414, NIGP01001415, NIGP01001416, NIGP01001417, NIGP01001426, NIGP01001431, NIGP01001438, NIGP01001442, NIGP01001450, NIGP01001455, NIGP01001458, NIGP01001463, NIGP01001464, NIGP01001468, NIGP01001469, NIGP01001476, NIGP01001477, NIGP01001478, NIGP01001480, NIGP01001482, NIGP01001497, NIGP01001501, NIGP01001506, NIGP01001507, NIGP01001509, NIGP01001516, NIGP01001519, NIGP01001520, NIGP01001522, NIGP01001530, NIGP01001531, NIGP01001534, NIGP01001536, NIGP01001546, NIGP01001547, NIGP01001549, NIGP01001551, NIGP01001553, NIGP01001557, NIGP01001562, NIGP01001568, NIGP01001572, NIGP01001573, NIGP01001574, NIGP01001575, NIGP01001576, NIGP01001578, NIGP01001580, NIGP01001587, NIGP01001589, NIGP01001591, NIGP01001593, NIGP01001597, NIGP01001599, NIGP01001603, NIGP01001614, NIGP01001615, NIGP01001617, NIGP01001618, NIGP01001622, NIGP01001624, NIGP01001630, NIGP01001639, NIGP01001640, NIGP01001641, NIGP01001645, NIGP01001646, NIGP01001648, NIGP01001661, NIGP01001671, NIGP01001672, NIGP01001675, NIGP01001676, NIGP01001678, NIGP01001682, NIGP01001690, NIGP01001692, NIGP01001696, NIGP01001699, NIGP01001702, NIGP01001703, NIGP01001705, NIGP01001706, NIGP01001711, NIGP01001712, NIGP01001713, NIGP01001717, NIGP01001719, NIGP01001726, NIGP01001727, NIGP01001728, NIGP01001729, NIGP01001730, NIGP01001732, NIGP01001736, NIGP01001738, NIGP01001743, NIGP01001749, NIGP01001750, NIGP01001752, NIGP01001755, NIGP01001756, NIGP01001758, NIGP01001766, NIGP01001767, NIGP01001769, NIGP01001770, NIGP01001774, NIGP01001784, NIGP01001786, NIGP01001788, NIGP01001789, NIGP01001800, NIGP01001801, NIGP01001810, NIGP01001811, NIGP01001816, NIGP01001817, NIGP01001818, NIGP01001819, NIGP01

## Warning in .Seqinfo.mergexy(x, y): Each of the 2 combined objects has sequence levels not in the other:
##   - in 'x': NIGP01000081, NIGP01000723, NIGP01001312, NIGP01001552, NIGP01001579, NIGP01001581, NIGP01001642, NIGP01001658, NIGP01001735, NIGP01001852, NIGP01002067, NIGP01000332, NIGP01000588, NIGP01001651, NIGP01001665, NIGP01001990, NIGP01000226, NIGP01000358, NIGP01000825, NIGP01000917, NIGP01000946, NIGP01001002, NIGP01001056, NIGP01001101, NIGP01001349, NIGP01001814, NIGP01002025, NIGP01001900
##   - in 'y': NIGP01000006, NIGP01000009, NIGP01000010, NIGP01000015, NIGP01000025, NIGP01000028, NIGP01000033, NIGP01000036, NIGP01000043, NIGP01000049, NIGP01000051, NIGP01000052, NIGP01000055, NIGP01000056, NIGP01000062, NIGP01000063, NIGP01000064, NIGP01000073, NIGP01000075, NIGP01000076, NIGP01000077, NIGP01000079, NIGP01000080, NIGP01000083, NIGP01000087, NIGP01000094, NIGP01000096, NIGP01000101, NIGP01000102, NIGP01000104, NIGP01000106, NIGP01000107, NIGP01000110, NIGP01000115, NIGP01000116, NIGP01000117, NIGP01000121, NIGP01000123, NIGP01000124, NIGP01000125, NIGP01000129, NIGP01000130, NIGP01000133, NIGP01000134, NIGP01000137, NIGP01000139, NIGP01000144, NIGP01000149, NIGP01000156, NIGP01000158, NIGP01000159, NIGP01000172, NIGP01000177, NIGP01000180, NIGP01000183, NIGP01000184, NIGP01000185, NIGP01000186, NIGP01000187, NIGP01000188, NIGP01000189, NIGP01000197, NIGP01000198, NIGP01000200, NIGP01000201, NIGP01000203, NIGP01000204, NIGP01000205, NIGP01000206, NIGP01000207, NIGP01000210, NIGP01000212, NIGP01000213, NIGP01000216, NIGP01000218, NIGP01000220, NIGP01000221, NIGP01000222, NIGP01000223, NIGP01000225, NIGP01000228, NIGP01000230, NIGP01000236, NIGP01000238, NIGP01000239, NIGP01000240, NIGP01000244, NIGP01000247, NIGP01000248, NIGP01000250, NIGP01000253, NIGP01000254, NIGP01000256, NIGP01000257, NIGP01000262, NIGP01000263, NIGP01000265, NIGP01000267, NIGP01000270, NIGP01000280, NIGP01000281, NIGP01000288, NIGP01000290, NIGP01000292, NIGP01000294, NIGP01000295, NIGP01000297, NIGP01000298, NIGP01000309, NIGP01000310, NIGP01000312, NIGP01000315, NIGP01000319, NIGP01000324, NIGP01000325, NIGP01000329, NIGP01000330, NIGP01000345, NIGP01000349, NIGP01000351, NIGP01000352, NIGP01000356, NIGP01000357, NIGP01000359, NIGP01000363, NIGP01000365, NIGP01000371, NIGP01000374, NIGP01000375, NIGP01000376, NIGP01000377, NIGP01000379, NIGP01000383, NIGP01000386, NIGP01000396, NIGP01000397, NIGP01000407, NIGP01000410, NIGP01000412, NIGP01000413, NIGP01000415, NIGP01000417, NIGP01000421, NIGP01000422, NIGP01000424, NIGP01000428, NIGP01000430, NIGP01000432, NIGP01000434, NIGP01000437, NIGP01000440, NIGP01000441, NIGP01000445, NIGP01000451, NIGP01000452, NIGP01000461, NIGP01000463, NIGP01000465, NIGP01000466, NIGP01000475, NIGP01000481, NIGP01000482, NIGP01000489, NIGP01000490, NIGP01000495, NIGP01000497, NIGP01000501, NIGP01000507, NIGP01000514, NIGP01000515, NIGP01000518, NIGP01000519, NIGP01000520, NIGP01000521, NIGP01000524, NIGP01000525, NIGP01000530, NIGP01000531, NIGP01000532, NIGP01000535, NIGP01000536, NIGP01000537, NIGP01000539, NIGP01000541, NIGP01000552, NIGP01000553, NIGP01000556, NIGP01000557, NIGP01000560, NIGP01000561, NIGP01000566, NIGP01000569, NIGP01000570, NIGP01000571, NIGP01000573, NIGP01000574, NIGP01000579, NIGP01000590, NIGP01000591, NIGP01000596, NIGP01000598, NIGP01000599, NIGP01000600, NIGP01000601, NIGP01000602, NIGP01000604, NIGP01000608, NIGP01000610, NIGP01000612, NIGP01000622, NIGP01000627, NIGP01000630, NIGP01000633, NIGP01000634, NIGP01000635, NIGP01000639, NIGP01000640, NIGP01000648, NIGP01000649, NIGP01000650, NIGP01000651, NIGP01000657, NIGP01000658, NIGP01000660, NIGP01000662, NIGP01000665, NIGP01000666, NIGP01000667, NIGP01000669, NIGP01000673, NIGP01000676, NIGP01000681, NIGP01000684, NIGP01000686, NIGP01000687, NIGP01000690, NIGP01000692, NIGP01000695, NIGP01000699, NIGP01000702, NIGP01000703, NIGP01000706, NIGP01000709, NIGP01000710, NIGP01000712, NIGP01000714, NIGP01000715, NIGP01000716, NIGP01000718, NIGP01000721, NIGP01000724, NIGP01000727, NIGP01000730, NIGP01000734, NIGP01000737, NIGP01000740, NIGP01000745, NIGP01000751, NIGP01000754, NIGP01000756, NIGP01000760, NIGP01000761, NIGP01000775, NIGP01000778, NIGP01000779, NIGP01000781, NIGP01000782, NIGP01000783, NIGP01000791, NIGP01000799, NIGP01000802, NIGP01000806, NIGP01000807, NIGP01000811, NIGP01000812, NIGP01000813, NIGP01000818, NIGP01000821, NIGP01000822, NIGP01000823, NIGP01000826, NIGP01000827, NIGP01000835, NIGP01000843, NIGP01000846, NIGP01000847, NIGP01000848, NIGP01000849, NIGP01000850, NIGP01000856, NIGP01000859, NIGP01000860, NIGP01000861, NIGP01000867, NIGP01000870, NIGP01000882, NIGP01000883, NIGP01000885, NIGP01000892, NIGP01000896, NIGP01000900, NIGP01000904, NIGP01000905, NIGP01000906, NIGP01000910, NIGP01000915, NIGP01000920, NIGP01000921, NIGP01000923, NIGP01000929, NIGP01000932, NIGP01000937, NIGP01000938, NIGP01000939, NIGP01000941, NIGP01000942, NIGP01000944, NIGP01000947, NIGP01000948, NIGP01000952, NIGP01000954, NIGP01000957, NIGP01000958, NIGP01000959, NIGP01000960, NIGP01000976, NIGP01000977, NIGP01000978, NIGP01000980, NIGP01000983, NIGP01000986, NIGP01000991, NIGP01000996, NIGP01001003, NIGP01001006, NIGP01001008, NIGP01001010, NIGP01001013, NIGP01001015, NIGP01001018, NIGP01001022, NIGP01001023, NIGP01001024, NIGP01001031, NIGP01001032, NIGP01001035, NIGP01001039, NIGP01001041, NIGP01001043, NIGP01001046, NIGP01001047, NIGP01001053, NIGP01001055, NIGP01001057, NIGP01001059, NIGP01001061, NIGP01001068, NIGP01001070, NIGP01001072, NIGP01001074, NIGP01001075, NIGP01001081, NIGP01001082, NIGP01001083, NIGP01001085, NIGP01001086, NIGP01001092, NIGP01001097, NIGP01001100, NIGP01001102, NIGP01001108, NIGP01001110, NIGP01001119, NIGP01001126, NIGP01001136, NIGP01001165, NIGP01001173, NIGP01001180, NIGP01001185, NIGP01001186, NIGP01001196, NIGP01001197, NIGP01001208, NIGP01001211, NIGP01001214, NIGP01001216, NIGP01001230, NIGP01001233, NIGP01001237, NIGP01001238, NIGP01001240, NIGP01001243, NIGP01001247, NIGP01001249, NIGP01001254, NIGP01001255, NIGP01001256, NIGP01001262, NIGP01001263, NIGP01001282, NIGP01001285, NIGP01001287, NIGP01001295, NIGP01001296, NIGP01001297, NIGP01001301, NIGP01001311, NIGP01001322, NIGP01001325, NIGP01001329, NIGP01001335, NIGP01001341, NIGP01001343, NIGP01001348, NIGP01001350, NIGP01001352, NIGP01001353, NIGP01001355, NIGP01001363, NIGP01001365, NIGP01001372, NIGP01001373, NIGP01001374, NIGP01001377, NIGP01001382, NIGP01001386, NIGP01001397, NIGP01001398, NIGP01001407, NIGP01001408, NIGP01001414, NIGP01001415, NIGP01001416, NIGP01001417, NIGP01001426, NIGP01001431, NIGP01001438, NIGP01001442, NIGP01001450, NIGP01001455, NIGP01001458, NIGP01001463, NIGP01001464, NIGP01001468, NIGP01001469, NIGP01001476, NIGP01001477, NIGP01001478, NIGP01001480, NIGP01001482, NIGP01001497, NIGP01001501, NIGP01001506, NIGP01001507, NIGP01001509, NIGP01001516, NIGP01001519, NIGP01001520, NIGP01001522, NIGP01001530, NIGP01001531, NIGP01001534, NIGP01001536, NIGP01001546, NIGP01001547, NIGP01001549, NIGP01001551, NIGP01001553, NIGP01001557, NIGP01001562, NIGP01001568, NIGP01001572, NIGP01001573, NIGP01001574, NIGP01001575, NIGP01001576, NIGP01001578, NIGP01001580, NIGP01001587, NIGP01001589, NIGP01001591, NIGP01001593, NIGP01001597, NIGP01001599, NIGP01001603, NIGP01001614, NIGP01001615, NIGP01001617, NIGP01001618, NIGP01001622, NIGP01001624, NIGP01001630, NIGP01001639, NIGP01001640, NIGP01001641, NIGP01001645, NIGP01001646, NIGP01001648, NIGP01001661, NIGP01001671, NIGP01001672, NIGP01001675, NIGP01001676, NIGP01001678, NIGP01001682, NIGP01001690, NIGP01001692, NIGP01001696, NIGP01001699, NIGP01001702, NIGP01001703, NIGP01001705, NIGP01001706, NIGP01001711, NIGP01001712, NIGP01001713, NIGP01001717, NIGP01001719, NIGP01001726, NIGP01001727, NIGP01001728, NIGP01001729, NIGP01001730, NIGP01001732, NIGP01001736, NIGP01001738, NIGP01001743, NIGP01001749, NIGP01001750, NIGP01001752, NIGP01001755, NIGP01001756, NIGP01001758, NIGP01001766, NIGP01001767, NIGP01001769, NIGP01001770, NIGP01001774, NIGP01001784, NIGP01001786, NIGP01001788, NIGP01001789, NIGP01001800, NIGP01001801, NIGP01001810, NIGP01001811, NIGP01001816, NIGP01001817, NIGP01001818, NIGP01001819, NIGP01

## >> calculating distance from peak to TSS...   2021-03-16 02:33:31 
## >> assigning genomic annotation...        2021-03-16 02:33:31

## Warning in .Seqinfo.mergexy(x, y): Each of the 2 combined objects has sequence levels not in the other:
##   - in 'x': NIGP01000081, NIGP01000723, NIGP01001312, NIGP01001552, NIGP01001579, NIGP01001581, NIGP01001642, NIGP01001658, NIGP01001735, NIGP01001852, NIGP01002067, NIGP01000332, NIGP01000588, NIGP01001651, NIGP01001665, NIGP01001990, NIGP01000226, NIGP01000358, NIGP01000825, NIGP01000917, NIGP01000946, NIGP01001002, NIGP01001056, NIGP01001101, NIGP01001349, NIGP01001814, NIGP01002025, NIGP01001900
##   - in 'y': NIGP01000006, NIGP01000009, NIGP01000010, NIGP01000015, NIGP01000025, NIGP01000028, NIGP01000033, NIGP01000036, NIGP01000043, NIGP01000049, NIGP01000051, NIGP01000052, NIGP01000055, NIGP01000056, NIGP01000062, NIGP01000063, NIGP01000064, NIGP01000073, NIGP01000075, NIGP01000076, NIGP01000077, NIGP01000079, NIGP01000080, NIGP01000083, NIGP01000087, NIGP01000094, NIGP01000096, NIGP01000101, NIGP01000102, NIGP01000104, NIGP01000106, NIGP01000107, NIGP01000110, NIGP01000115, NIGP01000116, NIGP01000117, NIGP01000121, NIGP01000123, NIGP01000124, NIGP01000125, NIGP01000129, NIGP01000130, NIGP01000133, NIGP01000134, NIGP01000137, NIGP01000139, NIGP01000144, NIGP01000149, NIGP01000156, NIGP01000158, NIGP01000159, NIGP01000172, NIGP01000177, NIGP01000180, NIGP01000183, NIGP01000184, NIGP01000185, NIGP01000186, NIGP01000187, NIGP01000188, NIGP01000189, NIGP01000197, NIGP01000198, NIGP01000200, NIGP01000201, NIGP01000203, NIGP01000204, NIGP01000205, NIGP01000206, NIGP01000207, NIGP01000210, NIGP01000212, NIGP01000213, NIGP01000216, NIGP01000218, NIGP01000220, NIGP01000221, NIGP01000222, NIGP01000223, NIGP01000225, NIGP01000228, NIGP01000230, NIGP01000236, NIGP01000238, NIGP01000239, NIGP01000240, NIGP01000244, NIGP01000247, NIGP01000248, NIGP01000250, NIGP01000253, NIGP01000254, NIGP01000256, NIGP01000257, NIGP01000262, NIGP01000263, NIGP01000265, NIGP01000267, NIGP01000270, NIGP01000280, NIGP01000281, NIGP01000288, NIGP01000290, NIGP01000292, NIGP01000294, NIGP01000295, NIGP01000297, NIGP01000298, NIGP01000309, NIGP01000310, NIGP01000312, NIGP01000315, NIGP01000319, NIGP01000324, NIGP01000325, NIGP01000329, NIGP01000330, NIGP01000345, NIGP01000349, NIGP01000351, NIGP01000352, NIGP01000356, NIGP01000357, NIGP01000359, NIGP01000363, NIGP01000365, NIGP01000371, NIGP01000374, NIGP01000375, NIGP01000376, NIGP01000377, NIGP01000379, NIGP01000383, NIGP01000386, NIGP01000396, NIGP01000397, NIGP01000407, NIGP01000410, NIGP01000412, NIGP01000413, NIGP01000415, NIGP01000417, NIGP01000421, NIGP01000422, NIGP01000424, NIGP01000428, NIGP01000430, NIGP01000432, NIGP01000434, NIGP01000437, NIGP01000440, NIGP01000441, NIGP01000445, NIGP01000451, NIGP01000452, NIGP01000461, NIGP01000463, NIGP01000465, NIGP01000466, NIGP01000475, NIGP01000481, NIGP01000482, NIGP01000489, NIGP01000490, NIGP01000495, NIGP01000497, NIGP01000501, NIGP01000507, NIGP01000514, NIGP01000515, NIGP01000518, NIGP01000519, NIGP01000520, NIGP01000521, NIGP01000524, NIGP01000525, NIGP01000530, NIGP01000531, NIGP01000532, NIGP01000535, NIGP01000536, NIGP01000537, NIGP01000539, NIGP01000541, NIGP01000552, NIGP01000553, NIGP01000556, NIGP01000557, NIGP01000560, NIGP01000561, NIGP01000566, NIGP01000569, NIGP01000570, NIGP01000571, NIGP01000573, NIGP01000574, NIGP01000579, NIGP01000590, NIGP01000591, NIGP01000596, NIGP01000598, NIGP01000599, NIGP01000600, NIGP01000601, NIGP01000602, NIGP01000604, NIGP01000608, NIGP01000610, NIGP01000612, NIGP01000622, NIGP01000627, NIGP01000630, NIGP01000633, NIGP01000634, NIGP01000635, NIGP01000639, NIGP01000640, NIGP01000648, NIGP01000649, NIGP01000650, NIGP01000651, NIGP01000657, NIGP01000658, NIGP01000660, NIGP01000662, NIGP01000665, NIGP01000666, NIGP01000667, NIGP01000669, NIGP01000673, NIGP01000676, NIGP01000681, NIGP01000684, NIGP01000686, NIGP01000687, NIGP01000690, NIGP01000692, NIGP01000695, NIGP01000699, NIGP01000702, NIGP01000703, NIGP01000706, NIGP01000709, NIGP01000710, NIGP01000712, NIGP01000714, NIGP01000715, NIGP01000716, NIGP01000718, NIGP01000721, NIGP01000724, NIGP01000727, NIGP01000730, NIGP01000734, NIGP01000737, NIGP01000740, NIGP01000745, NIGP01000751, NIGP01000754, NIGP01000756, NIGP01000760, NIGP01000761, NIGP01000775, NIGP01000778, NIGP01000779, NIGP01000781, NIGP01000782, NIGP01000783, NIGP01000791, NIGP01000799, NIGP01000802, NIGP01000806, NIGP01000807, NIGP01000811, NIGP01000812, NIGP01000813, NIGP01000818, NIGP01000821, NIGP01000822, NIGP01000823, NIGP01000826, NIGP01000827, NIGP01000835, NIGP01000843, NIGP01000846, NIGP01000847, NIGP01000848, NIGP01000849, NIGP01000850, NIGP01000856, NIGP01000859, NIGP01000860, NIGP01000861, NIGP01000867, NIGP01000870, NIGP01000882, NIGP01000883, NIGP01000885, NIGP01000892, NIGP01000896, NIGP01000900, NIGP01000904, NIGP01000905, NIGP01000906, NIGP01000910, NIGP01000915, NIGP01000920, NIGP01000921, NIGP01000923, NIGP01000929, NIGP01000932, NIGP01000937, NIGP01000938, NIGP01000939, NIGP01000941, NIGP01000942, NIGP01000944, NIGP01000947, NIGP01000948, NIGP01000952, NIGP01000954, NIGP01000957, NIGP01000958, NIGP01000959, NIGP01000960, NIGP01000976, NIGP01000977, NIGP01000978, NIGP01000980, NIGP01000983, NIGP01000986, NIGP01000991, NIGP01000996, NIGP01001003, NIGP01001006, NIGP01001008, NIGP01001010, NIGP01001013, NIGP01001015, NIGP01001018, NIGP01001022, NIGP01001023, NIGP01001024, NIGP01001031, NIGP01001032, NIGP01001035, NIGP01001039, NIGP01001041, NIGP01001043, NIGP01001046, NIGP01001047, NIGP01001053, NIGP01001055, NIGP01001057, NIGP01001059, NIGP01001061, NIGP01001068, NIGP01001070, NIGP01001072, NIGP01001074, NIGP01001075, NIGP01001081, NIGP01001082, NIGP01001083, NIGP01001085, NIGP01001086, NIGP01001092, NIGP01001097, NIGP01001100, NIGP01001102, NIGP01001108, NIGP01001110, NIGP01001119, NIGP01001126, NIGP01001136, NIGP01001165, NIGP01001173, NIGP01001180, NIGP01001185, NIGP01001186, NIGP01001196, NIGP01001197, NIGP01001208, NIGP01001211, NIGP01001214, NIGP01001216, NIGP01001230, NIGP01001233, NIGP01001237, NIGP01001238, NIGP01001240, NIGP01001243, NIGP01001247, NIGP01001249, NIGP01001254, NIGP01001255, NIGP01001256, NIGP01001262, NIGP01001263, NIGP01001282, NIGP01001285, NIGP01001287, NIGP01001295, NIGP01001296, NIGP01001297, NIGP01001301, NIGP01001311, NIGP01001322, NIGP01001325, NIGP01001329, NIGP01001335, NIGP01001341, NIGP01001343, NIGP01001348, NIGP01001350, NIGP01001352, NIGP01001353, NIGP01001355, NIGP01001363, NIGP01001365, NIGP01001372, NIGP01001373, NIGP01001374, NIGP01001377, NIGP01001382, NIGP01001386, NIGP01001397, NIGP01001398, NIGP01001407, NIGP01001408, NIGP01001414, NIGP01001415, NIGP01001416, NIGP01001417, NIGP01001426, NIGP01001431, NIGP01001438, NIGP01001442, NIGP01001450, NIGP01001455, NIGP01001458, NIGP01001463, NIGP01001464, NIGP01001468, NIGP01001469, NIGP01001476, NIGP01001477, NIGP01001478, NIGP01001480, NIGP01001482, NIGP01001497, NIGP01001501, NIGP01001506, NIGP01001507, NIGP01001509, NIGP01001516, NIGP01001519, NIGP01001520, NIGP01001522, NIGP01001530, NIGP01001531, NIGP01001534, NIGP01001536, NIGP01001546, NIGP01001547, NIGP01001549, NIGP01001551, NIGP01001553, NIGP01001557, NIGP01001562, NIGP01001568, NIGP01001572, NIGP01001573, NIGP01001574, NIGP01001575, NIGP01001576, NIGP01001578, NIGP01001580, NIGP01001587, NIGP01001589, NIGP01001591, NIGP01001593, NIGP01001597, NIGP01001599, NIGP01001603, NIGP01001614, NIGP01001615, NIGP01001617, NIGP01001618, NIGP01001622, NIGP01001624, NIGP01001630, NIGP01001639, NIGP01001640, NIGP01001641, NIGP01001645, NIGP01001646, NIGP01001648, NIGP01001661, NIGP01001671, NIGP01001672, NIGP01001675, NIGP01001676, NIGP01001678, NIGP01001682, NIGP01001690, NIGP01001692, NIGP01001696, NIGP01001699, NIGP01001702, NIGP01001703, NIGP01001705, NIGP01001706, NIGP01001711, NIGP01001712, NIGP01001713, NIGP01001717, NIGP01001719, NIGP01001726, NIGP01001727, NIGP01001728, NIGP01001729, NIGP01001730, NIGP01001732, NIGP01001736, NIGP01001738, NIGP01001743, NIGP01001749, NIGP01001750, NIGP01001752, NIGP01001755, NIGP01001756, NIGP01001758, NIGP01001766, NIGP01001767, NIGP01001769, NIGP01001770, NIGP01001774, NIGP01001784, NIGP01001786, NIGP01001788, NIGP01001789, NIGP01001800, NIGP01001801, NIGP01001810, NIGP01001811, NIGP01001816, NIGP01001817, NIGP01001818, NIGP01001819, NIGP01

## Warning in .Seqinfo.mergexy(x, y): Each of the 2 combined objects has sequence levels not in the other:
##   - in 'x': NIGP01000081, NIGP01000723, NIGP01001312, NIGP01001552, NIGP01001579, NIGP01001581, NIGP01001642, NIGP01001658, NIGP01001735, NIGP01001852, NIGP01002067, NIGP01000332, NIGP01000588, NIGP01001651, NIGP01001665, NIGP01001990, NIGP01000226, NIGP01000358, NIGP01000825, NIGP01000917, NIGP01000946, NIGP01001002, NIGP01001056, NIGP01001101, NIGP01001349, NIGP01001814, NIGP01002025, NIGP01001900
##   - in 'y': NIGP01000006, NIGP01000009, NIGP01000010, NIGP01000015, NIGP01000025, NIGP01000028, NIGP01000033, NIGP01000036, NIGP01000043, NIGP01000049, NIGP01000051, NIGP01000052, NIGP01000055, NIGP01000056, NIGP01000062, NIGP01000063, NIGP01000064, NIGP01000073, NIGP01000075, NIGP01000076, NIGP01000077, NIGP01000079, NIGP01000080, NIGP01000083, NIGP01000087, NIGP01000094, NIGP01000096, NIGP01000101, NIGP01000102, NIGP01000104, NIGP01000106, NIGP01000107, NIGP01000110, NIGP01000115, NIGP01000116, NIGP01000117, NIGP01000121, NIGP01000123, NIGP01000124, NIGP01000125, NIGP01000129, NIGP01000130, NIGP01000133, NIGP01000134, NIGP01000137, NIGP01000139, NIGP01000144, NIGP01000149, NIGP01000156, NIGP01000158, NIGP01000159, NIGP01000172, NIGP01000177, NIGP01000180, NIGP01000183, NIGP01000184, NIGP01000185, NIGP01000186, NIGP01000187, NIGP01000188, NIGP01000189, NIGP01000197, NIGP01000198, NIGP01000200, NIGP01000201, NIGP01000203, NIGP01000204, NIGP01000205, NIGP01000206, NIGP01000207, NIGP01000210, NIGP01000212, NIGP01000213, NIGP01000216, NIGP01000218, NIGP01000220, NIGP01000221, NIGP01000222, NIGP01000223, NIGP01000225, NIGP01000228, NIGP01000230, NIGP01000236, NIGP01000238, NIGP01000239, NIGP01000240, NIGP01000244, NIGP01000247, NIGP01000248, NIGP01000250, NIGP01000253, NIGP01000254, NIGP01000256, NIGP01000257, NIGP01000262, NIGP01000263, NIGP01000265, NIGP01000267, NIGP01000270, NIGP01000280, NIGP01000281, NIGP01000288, NIGP01000290, NIGP01000292, NIGP01000294, NIGP01000295, NIGP01000297, NIGP01000298, NIGP01000309, NIGP01000310, NIGP01000312, NIGP01000315, NIGP01000319, NIGP01000324, NIGP01000325, NIGP01000329, NIGP01000330, NIGP01000345, NIGP01000349, NIGP01000351, NIGP01000352, NIGP01000356, NIGP01000357, NIGP01000359, NIGP01000363, NIGP01000365, NIGP01000371, NIGP01000374, NIGP01000375, NIGP01000376, NIGP01000377, NIGP01000379, NIGP01000383, NIGP01000386, NIGP01000396, NIGP01000397, NIGP01000407, NIGP01000410, NIGP01000412, NIGP01000413, NIGP01000415, NIGP01000417, NIGP01000421, NIGP01000422, NIGP01000424, NIGP01000428, NIGP01000430, NIGP01000432, NIGP01000434, NIGP01000437, NIGP01000440, NIGP01000441, NIGP01000445, NIGP01000451, NIGP01000452, NIGP01000461, NIGP01000463, NIGP01000465, NIGP01000466, NIGP01000475, NIGP01000481, NIGP01000482, NIGP01000489, NIGP01000490, NIGP01000495, NIGP01000497, NIGP01000501, NIGP01000507, NIGP01000514, NIGP01000515, NIGP01000518, NIGP01000519, NIGP01000520, NIGP01000521, NIGP01000524, NIGP01000525, NIGP01000530, NIGP01000531, NIGP01000532, NIGP01000535, NIGP01000536, NIGP01000537, NIGP01000539, NIGP01000541, NIGP01000552, NIGP01000553, NIGP01000556, NIGP01000557, NIGP01000560, NIGP01000561, NIGP01000566, NIGP01000569, NIGP01000570, NIGP01000571, NIGP01000573, NIGP01000574, NIGP01000579, NIGP01000590, NIGP01000591, NIGP01000596, NIGP01000598, NIGP01000599, NIGP01000600, NIGP01000601, NIGP01000602, NIGP01000604, NIGP01000608, NIGP01000610, NIGP01000612, NIGP01000622, NIGP01000627, NIGP01000630, NIGP01000633, NIGP01000634, NIGP01000635, NIGP01000639, NIGP01000640, NIGP01000648, NIGP01000649, NIGP01000650, NIGP01000651, NIGP01000657, NIGP01000658, NIGP01000660, NIGP01000662, NIGP01000665, NIGP01000666, NIGP01000667, NIGP01000669, NIGP01000673, NIGP01000676, NIGP01000681, NIGP01000684, NIGP01000686, NIGP01000687, NIGP01000690, NIGP01000692, NIGP01000695, NIGP01000699, NIGP01000702, NIGP01000703, NIGP01000706, NIGP01000709, NIGP01000710, NIGP01000712, NIGP01000714, NIGP01000715, NIGP01000716, NIGP01000718, NIGP01000721, NIGP01000724, NIGP01000727, NIGP01000730, NIGP01000734, NIGP01000737, NIGP01000740, NIGP01000745, NIGP01000751, NIGP01000754, NIGP01000756, NIGP01000760, NIGP01000761, NIGP01000775, NIGP01000778, NIGP01000779, NIGP01000781, NIGP01000782, NIGP01000783, NIGP01000791, NIGP01000799, NIGP01000802, NIGP01000806, NIGP01000807, NIGP01000811, NIGP01000812, NIGP01000813, NIGP01000818, NIGP01000821, NIGP01000822, NIGP01000823, NIGP01000826, NIGP01000827, NIGP01000835, NIGP01000843, NIGP01000846, NIGP01000847, NIGP01000848, NIGP01000849, NIGP01000850, NIGP01000856, NIGP01000859, NIGP01000860, NIGP01000861, NIGP01000867, NIGP01000870, NIGP01000882, NIGP01000883, NIGP01000885, NIGP01000892, NIGP01000896, NIGP01000900, NIGP01000904, NIGP01000905, NIGP01000906, NIGP01000910, NIGP01000915, NIGP01000920, NIGP01000921, NIGP01000923, NIGP01000929, NIGP01000932, NIGP01000937, NIGP01000938, NIGP01000939, NIGP01000941, NIGP01000942, NIGP01000944, NIGP01000947, NIGP01000948, NIGP01000952, NIGP01000954, NIGP01000957, NIGP01000958, NIGP01000959, NIGP01000960, NIGP01000976, NIGP01000977, NIGP01000978, NIGP01000980, NIGP01000983, NIGP01000986, NIGP01000991, NIGP01000996, NIGP01001003, NIGP01001006, NIGP01001008, NIGP01001010, NIGP01001013, NIGP01001015, NIGP01001018, NIGP01001022, NIGP01001023, NIGP01001024, NIGP01001031, NIGP01001032, NIGP01001035, NIGP01001039, NIGP01001041, NIGP01001043, NIGP01001046, NIGP01001047, NIGP01001053, NIGP01001055, NIGP01001057, NIGP01001059, NIGP01001061, NIGP01001068, NIGP01001070, NIGP01001072, NIGP01001074, NIGP01001075, NIGP01001081, NIGP01001082, NIGP01001083, NIGP01001085, NIGP01001086, NIGP01001092, NIGP01001097, NIGP01001100, NIGP01001102, NIGP01001108, NIGP01001110, NIGP01001119, NIGP01001126, NIGP01001136, NIGP01001165, NIGP01001173, NIGP01001180, NIGP01001185, NIGP01001186, NIGP01001196, NIGP01001197, NIGP01001208, NIGP01001211, NIGP01001214, NIGP01001216, NIGP01001230, NIGP01001233, NIGP01001237, NIGP01001238, NIGP01001240, NIGP01001243, NIGP01001247, NIGP01001249, NIGP01001254, NIGP01001255, NIGP01001256, NIGP01001262, NIGP01001263, NIGP01001282, NIGP01001285, NIGP01001287, NIGP01001295, NIGP01001296, NIGP01001297, NIGP01001301, NIGP01001311, NIGP01001322, NIGP01001325, NIGP01001329, NIGP01001335, NIGP01001341, NIGP01001343, NIGP01001348, NIGP01001350, NIGP01001352, NIGP01001353, NIGP01001355, NIGP01001363, NIGP01001365, NIGP01001372, NIGP01001373, NIGP01001374, NIGP01001377, NIGP01001382, NIGP01001386, NIGP01001397, NIGP01001398, NIGP01001407, NIGP01001408, NIGP01001414, NIGP01001415, NIGP01001416, NIGP01001417, NIGP01001426, NIGP01001431, NIGP01001438, NIGP01001442, NIGP01001450, NIGP01001455, NIGP01001458, NIGP01001463, NIGP01001464, NIGP01001468, NIGP01001469, NIGP01001476, NIGP01001477, NIGP01001478, NIGP01001480, NIGP01001482, NIGP01001497, NIGP01001501, NIGP01001506, NIGP01001507, NIGP01001509, NIGP01001516, NIGP01001519, NIGP01001520, NIGP01001522, NIGP01001530, NIGP01001531, NIGP01001534, NIGP01001536, NIGP01001546, NIGP01001547, NIGP01001549, NIGP01001551, NIGP01001553, NIGP01001557, NIGP01001562, NIGP01001568, NIGP01001572, NIGP01001573, NIGP01001574, NIGP01001575, NIGP01001576, NIGP01001578, NIGP01001580, NIGP01001587, NIGP01001589, NIGP01001591, NIGP01001593, NIGP01001597, NIGP01001599, NIGP01001603, NIGP01001614, NIGP01001615, NIGP01001617, NIGP01001618, NIGP01001622, NIGP01001624, NIGP01001630, NIGP01001639, NIGP01001640, NIGP01001641, NIGP01001645, NIGP01001646, NIGP01001648, NIGP01001661, NIGP01001671, NIGP01001672, NIGP01001675, NIGP01001676, NIGP01001678, NIGP01001682, NIGP01001690, NIGP01001692, NIGP01001696, NIGP01001699, NIGP01001702, NIGP01001703, NIGP01001705, NIGP01001706, NIGP01001711, NIGP01001712, NIGP01001713, NIGP01001717, NIGP01001719, NIGP01001726, NIGP01001727, NIGP01001728, NIGP01001729, NIGP01001730, NIGP01001732, NIGP01001736, NIGP01001738, NIGP01001743, NIGP01001749, NIGP01001750, NIGP01001752, NIGP01001755, NIGP01001756, NIGP01001758, NIGP01001766, NIGP01001767, NIGP01001769, NIGP01001770, NIGP01001774, NIGP01001784, NIGP01001786, NIGP01001788, NIGP01001789, NIGP01001800, NIGP01001801, NIGP01001810, NIGP01001811, NIGP01001816, NIGP01001817, NIGP01001818, NIGP01001819, NIGP01

## Warning in .Seqinfo.mergexy(x, y): Each of the 2 combined objects has sequence levels not in the other:
##   - in 'x': NIGP01000081, NIGP01000723, NIGP01001312, NIGP01001552, NIGP01001579, NIGP01001581, NIGP01001642, NIGP01001658, NIGP01001735, NIGP01001852, NIGP01002067, NIGP01000332, NIGP01000588, NIGP01001651, NIGP01001665, NIGP01001990, NIGP01000226, NIGP01000358, NIGP01000825, NIGP01000917, NIGP01000946, NIGP01001002, NIGP01001056, NIGP01001101, NIGP01001349, NIGP01001814, NIGP01002025, NIGP01001900
##   - in 'y': NIGP01000006, NIGP01000009, NIGP01000010, NIGP01000015, NIGP01000025, NIGP01000028, NIGP01000033, NIGP01000036, NIGP01000043, NIGP01000049, NIGP01000051, NIGP01000052, NIGP01000055, NIGP01000056, NIGP01000062, NIGP01000063, NIGP01000064, NIGP01000073, NIGP01000075, NIGP01000076, NIGP01000077, NIGP01000079, NIGP01000080, NIGP01000083, NIGP01000087, NIGP01000094, NIGP01000096, NIGP01000101, NIGP01000102, NIGP01000104, NIGP01000106, NIGP01000107, NIGP01000110, NIGP01000115, NIGP01000116, NIGP01000117, NIGP01000121, NIGP01000123, NIGP01000124, NIGP01000125, NIGP01000129, NIGP01000130, NIGP01000133, NIGP01000134, NIGP01000137, NIGP01000139, NIGP01000144, NIGP01000149, NIGP01000156, NIGP01000158, NIGP01000159, NIGP01000172, NIGP01000177, NIGP01000180, NIGP01000183, NIGP01000184, NIGP01000185, NIGP01000186, NIGP01000187, NIGP01000188, NIGP01000189, NIGP01000197, NIGP01000198, NIGP01000200, NIGP01000201, NIGP01000203, NIGP01000204, NIGP01000205, NIGP01000206, NIGP01000207, NIGP01000210, NIGP01000212, NIGP01000213, NIGP01000216, NIGP01000218, NIGP01000220, NIGP01000221, NIGP01000222, NIGP01000223, NIGP01000225, NIGP01000228, NIGP01000230, NIGP01000236, NIGP01000238, NIGP01000239, NIGP01000240, NIGP01000244, NIGP01000247, NIGP01000248, NIGP01000250, NIGP01000253, NIGP01000254, NIGP01000256, NIGP01000257, NIGP01000262, NIGP01000263, NIGP01000265, NIGP01000267, NIGP01000270, NIGP01000280, NIGP01000281, NIGP01000288, NIGP01000290, NIGP01000292, NIGP01000294, NIGP01000295, NIGP01000297, NIGP01000298, NIGP01000309, NIGP01000310, NIGP01000312, NIGP01000315, NIGP01000319, NIGP01000324, NIGP01000325, NIGP01000329, NIGP01000330, NIGP01000345, NIGP01000349, NIGP01000351, NIGP01000352, NIGP01000356, NIGP01000357, NIGP01000359, NIGP01000363, NIGP01000365, NIGP01000371, NIGP01000374, NIGP01000375, NIGP01000376, NIGP01000377, NIGP01000379, NIGP01000383, NIGP01000386, NIGP01000396, NIGP01000397, NIGP01000407, NIGP01000410, NIGP01000412, NIGP01000413, NIGP01000415, NIGP01000417, NIGP01000421, NIGP01000422, NIGP01000424, NIGP01000428, NIGP01000430, NIGP01000432, NIGP01000434, NIGP01000437, NIGP01000440, NIGP01000441, NIGP01000445, NIGP01000451, NIGP01000452, NIGP01000461, NIGP01000463, NIGP01000465, NIGP01000466, NIGP01000475, NIGP01000481, NIGP01000482, NIGP01000489, NIGP01000490, NIGP01000495, NIGP01000497, NIGP01000501, NIGP01000507, NIGP01000514, NIGP01000515, NIGP01000518, NIGP01000519, NIGP01000520, NIGP01000521, NIGP01000524, NIGP01000525, NIGP01000530, NIGP01000531, NIGP01000532, NIGP01000535, NIGP01000536, NIGP01000537, NIGP01000539, NIGP01000541, NIGP01000552, NIGP01000553, NIGP01000556, NIGP01000557, NIGP01000560, NIGP01000561, NIGP01000566, NIGP01000569, NIGP01000570, NIGP01000571, NIGP01000573, NIGP01000574, NIGP01000579, NIGP01000590, NIGP01000591, NIGP01000596, NIGP01000598, NIGP01000599, NIGP01000600, NIGP01000601, NIGP01000602, NIGP01000604, NIGP01000608, NIGP01000610, NIGP01000612, NIGP01000622, NIGP01000627, NIGP01000630, NIGP01000633, NIGP01000634, NIGP01000635, NIGP01000639, NIGP01000640, NIGP01000648, NIGP01000649, NIGP01000650, NIGP01000651, NIGP01000657, NIGP01000658, NIGP01000660, NIGP01000662, NIGP01000665, NIGP01000666, NIGP01000667, NIGP01000669, NIGP01000673, NIGP01000676, NIGP01000681, NIGP01000684, NIGP01000686, NIGP01000687, NIGP01000690, NIGP01000692, NIGP01000695, NIGP01000699, NIGP01000702, NIGP01000703, NIGP01000706, NIGP01000709, NIGP01000710, NIGP01000712, NIGP01000714, NIGP01000715, NIGP01000716, NIGP01000718, NIGP01000721, NIGP01000724, NIGP01000727, NIGP01000730, NIGP01000734, NIGP01000737, NIGP01000740, NIGP01000745, NIGP01000751, NIGP01000754, NIGP01000756, NIGP01000760, NIGP01000761, NIGP01000775, NIGP01000778, NIGP01000779, NIGP01000781, NIGP01000782, NIGP01000783, NIGP01000791, NIGP01000799, NIGP01000802, NIGP01000806, NIGP01000807, NIGP01000811, NIGP01000812, NIGP01000813, NIGP01000818, NIGP01000821, NIGP01000822, NIGP01000823, NIGP01000826, NIGP01000827, NIGP01000835, NIGP01000843, NIGP01000846, NIGP01000847, NIGP01000848, NIGP01000849, NIGP01000850, NIGP01000856, NIGP01000859, NIGP01000860, NIGP01000861, NIGP01000867, NIGP01000870, NIGP01000882, NIGP01000883, NIGP01000885, NIGP01000892, NIGP01000896, NIGP01000900, NIGP01000904, NIGP01000905, NIGP01000906, NIGP01000910, NIGP01000915, NIGP01000920, NIGP01000921, NIGP01000923, NIGP01000929, NIGP01000932, NIGP01000937, NIGP01000938, NIGP01000939, NIGP01000941, NIGP01000942, NIGP01000944, NIGP01000947, NIGP01000948, NIGP01000952, NIGP01000954, NIGP01000957, NIGP01000958, NIGP01000959, NIGP01000960, NIGP01000976, NIGP01000977, NIGP01000978, NIGP01000980, NIGP01000983, NIGP01000986, NIGP01000991, NIGP01000996, NIGP01001003, NIGP01001006, NIGP01001008, NIGP01001010, NIGP01001013, NIGP01001015, NIGP01001018, NIGP01001022, NIGP01001023, NIGP01001024, NIGP01001031, NIGP01001032, NIGP01001035, NIGP01001039, NIGP01001041, NIGP01001043, NIGP01001046, NIGP01001047, NIGP01001053, NIGP01001055, NIGP01001057, NIGP01001059, NIGP01001061, NIGP01001068, NIGP01001070, NIGP01001072, NIGP01001074, NIGP01001075, NIGP01001081, NIGP01001082, NIGP01001083, NIGP01001085, NIGP01001086, NIGP01001092, NIGP01001097, NIGP01001100, NIGP01001102, NIGP01001108, NIGP01001110, NIGP01001119, NIGP01001126, NIGP01001136, NIGP01001165, NIGP01001173, NIGP01001180, NIGP01001185, NIGP01001186, NIGP01001196, NIGP01001197, NIGP01001208, NIGP01001211, NIGP01001214, NIGP01001216, NIGP01001230, NIGP01001233, NIGP01001237, NIGP01001238, NIGP01001240, NIGP01001243, NIGP01001247, NIGP01001249, NIGP01001254, NIGP01001255, NIGP01001256, NIGP01001262, NIGP01001263, NIGP01001282, NIGP01001285, NIGP01001287, NIGP01001295, NIGP01001296, NIGP01001297, NIGP01001301, NIGP01001311, NIGP01001322, NIGP01001325, NIGP01001329, NIGP01001335, NIGP01001341, NIGP01001343, NIGP01001348, NIGP01001350, NIGP01001352, NIGP01001353, NIGP01001355, NIGP01001363, NIGP01001365, NIGP01001372, NIGP01001373, NIGP01001374, NIGP01001377, NIGP01001382, NIGP01001386, NIGP01001397, NIGP01001398, NIGP01001407, NIGP01001408, NIGP01001414, NIGP01001415, NIGP01001416, NIGP01001417, NIGP01001426, NIGP01001431, NIGP01001438, NIGP01001442, NIGP01001450, NIGP01001455, NIGP01001458, NIGP01001463, NIGP01001464, NIGP01001468, NIGP01001469, NIGP01001476, NIGP01001477, NIGP01001478, NIGP01001480, NIGP01001482, NIGP01001497, NIGP01001501, NIGP01001506, NIGP01001507, NIGP01001509, NIGP01001516, NIGP01001519, NIGP01001520, NIGP01001522, NIGP01001530, NIGP01001531, NIGP01001534, NIGP01001536, NIGP01001546, NIGP01001547, NIGP01001549, NIGP01001551, NIGP01001553, NIGP01001557, NIGP01001562, NIGP01001568, NIGP01001572, NIGP01001573, NIGP01001574, NIGP01001575, NIGP01001576, NIGP01001578, NIGP01001580, NIGP01001587, NIGP01001589, NIGP01001591, NIGP01001593, NIGP01001597, NIGP01001599, NIGP01001603, NIGP01001614, NIGP01001615, NIGP01001617, NIGP01001618, NIGP01001622, NIGP01001624, NIGP01001630, NIGP01001639, NIGP01001640, NIGP01001641, NIGP01001645, NIGP01001646, NIGP01001648, NIGP01001661, NIGP01001671, NIGP01001672, NIGP01001675, NIGP01001676, NIGP01001678, NIGP01001682, NIGP01001690, NIGP01001692, NIGP01001696, NIGP01001699, NIGP01001702, NIGP01001703, NIGP01001705, NIGP01001706, NIGP01001711, NIGP01001712, NIGP01001713, NIGP01001717, NIGP01001719, NIGP01001726, NIGP01001727, NIGP01001728, NIGP01001729, NIGP01001730, NIGP01001732, NIGP01001736, NIGP01001738, NIGP01001743, NIGP01001749, NIGP01001750, NIGP01001752, NIGP01001755, NIGP01001756, NIGP01001758, NIGP01001766, NIGP01001767, NIGP01001769, NIGP01001770, NIGP01001774, NIGP01001784, NIGP01001786, NIGP01001788, NIGP01001789, NIGP01001800, NIGP01001801, NIGP01001810, NIGP01001811, NIGP01001816, NIGP01001817, NIGP01001818, NIGP01001819, NIGP01

## Warning in .Seqinfo.mergexy(x, y): Each of the 2 combined objects has sequence levels not in the other:
##   - in 'x': NIGP01000081, NIGP01000723, NIGP01001312, NIGP01001552, NIGP01001579, NIGP01001581, NIGP01001642, NIGP01001658, NIGP01001735, NIGP01001852, NIGP01002067, NIGP01000332, NIGP01000588, NIGP01001651, NIGP01001665, NIGP01001990, NIGP01000226, NIGP01000358, NIGP01000825, NIGP01000917, NIGP01000946, NIGP01001002, NIGP01001056, NIGP01001101, NIGP01001349, NIGP01001814, NIGP01002025, NIGP01001900
##   - in 'y': NIGP01000006, NIGP01000009, NIGP01000010, NIGP01000015, NIGP01000025, NIGP01000028, NIGP01000033, NIGP01000036, NIGP01000043, NIGP01000049, NIGP01000051, NIGP01000052, NIGP01000055, NIGP01000056, NIGP01000062, NIGP01000063, NIGP01000064, NIGP01000073, NIGP01000075, NIGP01000076, NIGP01000077, NIGP01000079, NIGP01000080, NIGP01000083, NIGP01000087, NIGP01000094, NIGP01000096, NIGP01000101, NIGP01000102, NIGP01000104, NIGP01000106, NIGP01000107, NIGP01000110, NIGP01000115, NIGP01000116, NIGP01000117, NIGP01000121, NIGP01000123, NIGP01000124, NIGP01000125, NIGP01000129, NIGP01000130, NIGP01000133, NIGP01000134, NIGP01000137, NIGP01000139, NIGP01000144, NIGP01000149, NIGP01000156, NIGP01000158, NIGP01000159, NIGP01000172, NIGP01000177, NIGP01000180, NIGP01000183, NIGP01000184, NIGP01000185, NIGP01000186, NIGP01000187, NIGP01000188, NIGP01000189, NIGP01000197, NIGP01000198, NIGP01000200, NIGP01000201, NIGP01000203, NIGP01000204, NIGP01000205, NIGP01000206, NIGP01000207, NIGP01000210, NIGP01000212, NIGP01000213, NIGP01000216, NIGP01000218, NIGP01000220, NIGP01000221, NIGP01000222, NIGP01000223, NIGP01000225, NIGP01000228, NIGP01000230, NIGP01000236, NIGP01000238, NIGP01000239, NIGP01000240, NIGP01000244, NIGP01000247, NIGP01000248, NIGP01000250, NIGP01000253, NIGP01000254, NIGP01000256, NIGP01000257, NIGP01000262, NIGP01000263, NIGP01000265, NIGP01000267, NIGP01000270, NIGP01000280, NIGP01000281, NIGP01000288, NIGP01000290, NIGP01000292, NIGP01000294, NIGP01000295, NIGP01000297, NIGP01000298, NIGP01000309, NIGP01000310, NIGP01000312, NIGP01000315, NIGP01000319, NIGP01000324, NIGP01000325, NIGP01000329, NIGP01000330, NIGP01000345, NIGP01000349, NIGP01000351, NIGP01000352, NIGP01000356, NIGP01000357, NIGP01000359, NIGP01000363, NIGP01000365, NIGP01000371, NIGP01000374, NIGP01000375, NIGP01000376, NIGP01000377, NIGP01000379, NIGP01000383, NIGP01000386, NIGP01000396, NIGP01000397, NIGP01000407, NIGP01000410, NIGP01000412, NIGP01000413, NIGP01000415, NIGP01000417, NIGP01000421, NIGP01000422, NIGP01000424, NIGP01000428, NIGP01000430, NIGP01000432, NIGP01000434, NIGP01000437, NIGP01000440, NIGP01000441, NIGP01000445, NIGP01000451, NIGP01000452, NIGP01000461, NIGP01000463, NIGP01000465, NIGP01000466, NIGP01000475, NIGP01000481, NIGP01000482, NIGP01000489, NIGP01000490, NIGP01000495, NIGP01000497, NIGP01000501, NIGP01000507, NIGP01000514, NIGP01000515, NIGP01000518, NIGP01000519, NIGP01000520, NIGP01000521, NIGP01000524, NIGP01000525, NIGP01000530, NIGP01000531, NIGP01000532, NIGP01000535, NIGP01000536, NIGP01000537, NIGP01000539, NIGP01000541, NIGP01000552, NIGP01000553, NIGP01000556, NIGP01000557, NIGP01000560, NIGP01000561, NIGP01000566, NIGP01000569, NIGP01000570, NIGP01000571, NIGP01000573, NIGP01000574, NIGP01000579, NIGP01000590, NIGP01000591, NIGP01000596, NIGP01000598, NIGP01000599, NIGP01000600, NIGP01000601, NIGP01000602, NIGP01000604, NIGP01000608, NIGP01000610, NIGP01000612, NIGP01000622, NIGP01000627, NIGP01000630, NIGP01000633, NIGP01000634, NIGP01000635, NIGP01000639, NIGP01000640, NIGP01000648, NIGP01000649, NIGP01000650, NIGP01000651, NIGP01000657, NIGP01000658, NIGP01000660, NIGP01000662, NIGP01000665, NIGP01000666, NIGP01000667, NIGP01000669, NIGP01000673, NIGP01000676, NIGP01000681, NIGP01000684, NIGP01000686, NIGP01000687, NIGP01000690, NIGP01000692, NIGP01000695, NIGP01000699, NIGP01000702, NIGP01000703, NIGP01000706, NIGP01000709, NIGP01000710, NIGP01000712, NIGP01000714, NIGP01000715, NIGP01000716, NIGP01000718, NIGP01000721, NIGP01000724, NIGP01000727, NIGP01000730, NIGP01000734, NIGP01000737, NIGP01000740, NIGP01000745, NIGP01000751, NIGP01000754, NIGP01000756, NIGP01000760, NIGP01000761, NIGP01000775, NIGP01000778, NIGP01000779, NIGP01000781, NIGP01000782, NIGP01000783, NIGP01000791, NIGP01000799, NIGP01000802, NIGP01000806, NIGP01000807, NIGP01000811, NIGP01000812, NIGP01000813, NIGP01000818, NIGP01000821, NIGP01000822, NIGP01000823, NIGP01000826, NIGP01000827, NIGP01000835, NIGP01000843, NIGP01000846, NIGP01000847, NIGP01000848, NIGP01000849, NIGP01000850, NIGP01000856, NIGP01000859, NIGP01000860, NIGP01000861, NIGP01000867, NIGP01000870, NIGP01000882, NIGP01000883, NIGP01000885, NIGP01000892, NIGP01000896, NIGP01000900, NIGP01000904, NIGP01000905, NIGP01000906, NIGP01000910, NIGP01000915, NIGP01000920, NIGP01000921, NIGP01000923, NIGP01000929, NIGP01000932, NIGP01000937, NIGP01000938, NIGP01000939, NIGP01000941, NIGP01000942, NIGP01000944, NIGP01000947, NIGP01000948, NIGP01000952, NIGP01000954, NIGP01000957, NIGP01000958, NIGP01000959, NIGP01000960, NIGP01000976, NIGP01000977, NIGP01000978, NIGP01000980, NIGP01000983, NIGP01000986, NIGP01000991, NIGP01000996, NIGP01001003, NIGP01001006, NIGP01001008, NIGP01001010, NIGP01001013, NIGP01001015, NIGP01001018, NIGP01001022, NIGP01001023, NIGP01001024, NIGP01001031, NIGP01001032, NIGP01001035, NIGP01001039, NIGP01001041, NIGP01001043, NIGP01001046, NIGP01001047, NIGP01001053, NIGP01001055, NIGP01001057, NIGP01001059, NIGP01001061, NIGP01001068, NIGP01001070, NIGP01001072, NIGP01001074, NIGP01001075, NIGP01001081, NIGP01001082, NIGP01001083, NIGP01001085, NIGP01001086, NIGP01001092, NIGP01001097, NIGP01001100, NIGP01001102, NIGP01001108, NIGP01001110, NIGP01001119, NIGP01001126, NIGP01001136, NIGP01001165, NIGP01001173, NIGP01001180, NIGP01001185, NIGP01001186, NIGP01001196, NIGP01001197, NIGP01001208, NIGP01001211, NIGP01001214, NIGP01001216, NIGP01001230, NIGP01001233, NIGP01001237, NIGP01001238, NIGP01001240, NIGP01001243, NIGP01001247, NIGP01001249, NIGP01001254, NIGP01001255, NIGP01001256, NIGP01001262, NIGP01001263, NIGP01001282, NIGP01001285, NIGP01001287, NIGP01001295, NIGP01001296, NIGP01001297, NIGP01001301, NIGP01001311, NIGP01001322, NIGP01001325, NIGP01001329, NIGP01001335, NIGP01001341, NIGP01001343, NIGP01001348, NIGP01001350, NIGP01001352, NIGP01001353, NIGP01001355, NIGP01001363, NIGP01001365, NIGP01001372, NIGP01001373, NIGP01001374, NIGP01001377, NIGP01001382, NIGP01001386, NIGP01001397, NIGP01001398, NIGP01001407, NIGP01001408, NIGP01001414, NIGP01001415, NIGP01001416, NIGP01001417, NIGP01001426, NIGP01001431, NIGP01001438, NIGP01001442, NIGP01001450, NIGP01001455, NIGP01001458, NIGP01001463, NIGP01001464, NIGP01001468, NIGP01001469, NIGP01001476, NIGP01001477, NIGP01001478, NIGP01001480, NIGP01001482, NIGP01001497, NIGP01001501, NIGP01001506, NIGP01001507, NIGP01001509, NIGP01001516, NIGP01001519, NIGP01001520, NIGP01001522, NIGP01001530, NIGP01001531, NIGP01001534, NIGP01001536, NIGP01001546, NIGP01001547, NIGP01001549, NIGP01001551, NIGP01001553, NIGP01001557, NIGP01001562, NIGP01001568, NIGP01001572, NIGP01001573, NIGP01001574, NIGP01001575, NIGP01001576, NIGP01001578, NIGP01001580, NIGP01001587, NIGP01001589, NIGP01001591, NIGP01001593, NIGP01001597, NIGP01001599, NIGP01001603, NIGP01001614, NIGP01001615, NIGP01001617, NIGP01001618, NIGP01001622, NIGP01001624, NIGP01001630, NIGP01001639, NIGP01001640, NIGP01001641, NIGP01001645, NIGP01001646, NIGP01001648, NIGP01001661, NIGP01001671, NIGP01001672, NIGP01001675, NIGP01001676, NIGP01001678, NIGP01001682, NIGP01001690, NIGP01001692, NIGP01001696, NIGP01001699, NIGP01001702, NIGP01001703, NIGP01001705, NIGP01001706, NIGP01001711, NIGP01001712, NIGP01001713, NIGP01001717, NIGP01001719, NIGP01001726, NIGP01001727, NIGP01001728, NIGP01001729, NIGP01001730, NIGP01001732, NIGP01001736, NIGP01001738, NIGP01001743, NIGP01001749, NIGP01001750, NIGP01001752, NIGP01001755, NIGP01001756, NIGP01001758, NIGP01001766, NIGP01001767, NIGP01001769, NIGP01001770, NIGP01001774, NIGP01001784, NIGP01001786, NIGP01001788, NIGP01001789, NIGP01001800, NIGP01001801, NIGP01001810, NIGP01001811, NIGP01001816, NIGP01001817, NIGP01001818, NIGP01001819, NIGP01

## >> assigning chromosome lengths           2021-03-16 02:33:35 
## >> done...                    2021-03-16 02:33:35

plotAnnoPie(allanno)

Kathryn Rozen-Gagnon, Ji-Dung Luo and Thomas Carroll

Process standard/BrdU-CLIP libraries using CLIPflexR

Initial processing and quality control

Decompress fastq files

You can look at the quality of your data (wrapping FASTX’s quality stats)

Quality filter (wrapping FastX’s quality filter)

Fastx_collapser (wrapping FASTX’s fastx_collapser)

Follow these steps to process standard CLIP libraries

De-multiplex samples (wrapping FASTX’s barcode splitter)

Now remove the 5’ index, linker, and random barcode sequences (wrapping CTK’s stripBarcode.pl)

Now remove the 3’ linker (wrapping FASTX’s clipper)

These reads are now ready to map to your genome

Follow these steps to process BrdU-CLIP libraries

First strip random barcode sequences (UMI) from the 5’ end of reads (wrapping CTK’s stripBarcode.pl)

De-multiplex samples (wrapping FASTX’s barcode splitter)

Trim 3 “not G” (DDD) and index (6nt) from the 5’ end of reads (wrapping FASTX’s fastx_trimmer)

Fastx_clipper (wrapping FASTX’s fastx_clipper)

Build a Bowtie2 genome index and align to genome using Rbowtie2.

Convert to beds, find peaks, and build count matrix

Convert to beds

Make tag directories and findpeaks (wrapping HOMER’s MakeTagDirectory and findpeaks)

Make count matrix of peaks, starting with reading in HOMER peak files and making a matrix that indicated which peaks were called in each sample

The next step is to countFromBed to append the number of reads at each peak location, by sample

now you have your count matrix!

you can perform operations on sample groups to filter, visualize data, annotate your peaks, and more…