Pipeline

Tip

CPT3 can perform one click analysis or the following modules step by step:

tool

input files

main output file(s)

main application

Linker filter

Single/Paired-end fastq/gz files

clean PETs after linker filter

Perform Linker filter and qulaity contril for sequencing raw data

Alignment

clean fastq file

align sam/bam file

Perform Single-end alignment and perform paired

PET divide

aligned paired-end reads

valid pairs reads, self-ligation

Dividing aligned pair reads into different categories

Peak calling

valid pair reads

enrichment peak

Perform peak calling with MACS2 or SPP method

Loop calling

valid pair reads and peak results

valid loops

Perform loop calling analysis

CPT3 pipeline

An easy-to-use, auto-run package for ChIA-PET, HiChIP data analyses:

Raw reads:

java -jar ChIA-PET.jar --fastq1 test_long_1.fastq.gz --fastq2 test_long_2.fastq.gz \
    --minimum_linker_alignment_score 14 --GENOME_INDEX hg38.fa --GENOME_LENGTH 3E9 \
    --CHROM_SIZE_INFO ChIA-PET_Tool_V3/chromInfo/hg38.chromSize.txt \
    --CYTOBAND_DATA ChIA-PET_Tool_V3/chromInfo/hg19_cytoBandIdeo.txt --SPECIES 1 \
    --output test_long --prefix GM12878 --thread 8 --fastp fastp

Or clean reads:

java -jar ChIA-PET.jar --fastq1 test_long_1.fastq.gz --fastq2 test_long_2.fastq.gz \
    --minimum_linker_alignment_score 14 --GENOME_INDEX hg38.fa --GENOME_LENGTH 3E9 \
    --CHROM_SIZE_INFO ChIA-PET_Tool_V3/chromInfo/hg38.chromSize.txt \
    --CYTOBAND_DATA ChIA-PET_Tool_V3/chromInfo/hg19_cytoBandIdeo.txt --SPECIES 1 \
    --output test_long --prefix GM12878 --thread 8

With our test, we strong suggest --fastp paramater for analysis:

You can always see all available command-line options via --help:

$ java -jar ChIA-PET.jar --help

  • After the program runs successfully, a series of files with '-o' as prefix and report file will be generated in the output directory. Please refer to the doc for the specific output file and format details.

  • In addition, there will be an HTML report file containing basic information and statistical results of data analysis.

CPT3 pipeline main parameters

[ Main paramaters ]

System.out.println("Usage: java -jar <path of ChIA_PET.jar> [options]

Necessary options:

--fastq1

path of read1 fastq file

--fastq2

path of read2 fastq file

--autolinker

detect linker by our program, then no need provide --linker and --mode paramater.

--mode

mode of tool, 0: short read; 1: long read, need for ChIA-PET data

--linker

path of linker file, need for ChIA-PET mode

--fastp

fastp path, strong suggest for ChIA-PET data.

--skipheader

skip header N reads for detect linker, default 1000000.

--linkerreads

N reads used for detect linker, default 100000.

--hichip

Y(es) or N(o)[default] or O(nly print restriction site file without run other step), need for hichip data

--ligation_site

It can be the name of restriction enzyme, such as HindIII, MboI, DpnII, Bglii, Sau3AI, Hinf1, NlaIII, AluI or the site of enzyme digestion, A^AGCTT, ^GATC, ^GATC, A^GATCT, G^ANTC, CATG^, AG^CT or others. multipe restriction enzyme can be seperated by comma, such as G^ANTC,^GATC. restriction site with '^' and contains 'ATCG' without other character!!! if the genomic enzyme digestion file --restrictionsiteFile is provided, this parameter does not need to be provided. only needed for hichip data

--ResRomove

Y or N, whether remove PET in same restriction contig. default: Y

--restrictionsiteFile

restriction site file, can be genarated while has --ligation_site and without this paramater or provide restriction enzyme information with --ligation_site, we will automatically generate the file. only needed for hichip data

--genomefile

genome fasta file path, needed for with --ligation_site and without --restrictionsiteFile only needed for hichip data

--minfragsizeMinimum

restriction fragment length to consider, default 20

--maxfragsize

Maximum restriction fragment length to consider, default 1000000

--minInsertsize

Minimum restriction fragment skip of mapped reads to consider, default 1

--fqmode

single-end or paired-end (default), only required --fastq1 when single-end mode for ChIA-PET data

--minimum_linker_alignment_score

minimum alignment score, 14 default

--GENOME_INDEX

the path of BWA index

--GENOME_LENGTH

the number of base pairs in the whole genome

--CHROM_SIZE_INFO

the file that contains the length of each chromosome, example file is in ChIA-PET_Tool_V3/chrInfo, this is necessary for > step 2 analysis. Note. please make sure chromosome name in this file is same as name in genome file!!!

--CYTOBAND_DATA

the ideogram data used to plot intra-chromosomal peaks and interactions, example file is in ChIA-PET_Tool_V3/chrInfo

--SPECIES

1: human; 2: mouse; 3: others

Other options:

--start_step

start with which step, 1: linker filtering; 2: mapping to genome; 3: removing redundancy; 4: categorization of PETs; 5: peak calling; 6: interaction calling; 7: visualizing, default: 1

--stop_step

stop with which step, 1: linker filtering; 2: mapping to genome; 3: removing redundancy; 4: categorization of PETs; 5: peak calling; 6: interaction calling; 7: visualizing, default: 100, should be bigger than --start_step

--output

path of output, default: ChIA-PET_Tool_V3/output

--prefix

prefix of output files, default: out

--minimum_tag_length

minimum tag length, default: 18

--maximum_tag_length

maximum tag length, default: 1000

--minSecondBestScoreDiff

the score difference between the best-aligned and the second-best aligned linkers, default: 3

--output_data_with_ambiguous_linker_info

whether to print the linker-ambiguity PETs, 0: not print; 1: print, default: 1

--printreadID

write read ID to bedpe file, default: N

--printallreads

print all reads no matter strand, default: 0[print all]; 1, only print valid strand reads.

--search_all_linker

search all linkers in reads or just search one time, default: N

--thread

the number of threads used in linker filtering and mapping to genome, default: 1

--MAPPING_CUTOFF

cutoff of mapping quality score for filtering out low-quality or multiply-mapped reads, default: 20

--MERGE_DISTANCE

the distance limit to merge the PETs with similar mapping locations, default: 2

--SELF_LIGATION_CUFOFF

the distance threshold between self-ligation PETs and intra-chromosomal

inter-ligation PETs, default: 8000 for ChIA, and 1000 for HiChIP

--EXTENSION_LENGTH

the extension length from the location of each tag, default: 500, 1500 suggest for single-end mode

--MIN_COVERAGE_FOR_PEAK

the minimum coverage to define peak regions, default: 5

--PEAK_MODE

1: peak region mode, which takes all the overlapping PET regions above the coverage threshold as peak regions; 2: peak summit mode, which takes the highest coverage of overlapping regions as peak regions, default: 2

--MIN_DISTANCE_BETWEEN_PEAK

the minimum distance between two peaks, default: 500

--GENOME_COVERAGE_RATIO

the estimated proportion of the genome covered by the reads, default: 0.8

--PVALUE_CUTOFF_PEAK

p-value to filter peaks that are not statistically significant, default: 0.00001

--INPUT_ANCHOR_FILE

a file which contains user-specified anchors for interaction calling, default: null

--PVALUE_CUTOFF_INTERACTION | p-value to filter false positive interactions, default: 0.5

--zipbedpe

gzip bedpe related file, after analysis done. default: N. Y for gzip, N for not.

--zipsam

Convert sam file to bam, after analysis done. default: N

--deletesam

Delete sam files. default: N

--keeptemp

Keep temp sam and bedpe file. default: N

--map_ambiguous

Also mapping ambiguous reads without linker. default: N

--skipmap

Skip mapping read1 and read2, start from paired R1.sam and R2.sam, only valid in HiChIP mode now. default: N

--macs2

macs2 path, using macs2 callpeak to detect anchor peak with alignment file. default: N

--nomodel

macs2 parameter, Whether or not to build the shifting model in macs2. default: N

--shortestP

extend and keep shorest peak length longer than N for loop calling, suggest 1500, user can set 0 to skip this step. default: 1500

--shortestA

extend and keep shorest anchor length longer than N for loop calling, user can set 0 to skip this step. default: 0

--XOR_cluster

Whether keep loops if only one side of anchor is overlap with peak. default: N

--addcluster

Keep all regions with more than 2 count reads as potential anchor for calling loop. default: N. if peaks number of macs2 smaller than 10000, this paramater will work automaticly.

Output files

Output file format and details see "https://github.com/GuoliangLi-HZAU/ChIA-PET_Tool_V3#result-file".<br>

Output report details see "xxx" .<br>

Tip

For feature requests or bug reports please open an issue on github.