A script/tool for detecting postzygotic single-nucleotide mutations in human whole-genome sequencing data.
======Preparation
Make sure that you have installed the listed softwares, then added pre-installed softwares and the directory /your/MosaicHunter/directory/Tools in your PATH
In order to generate essential reference data and compile c, c++, java scripts, you should run this command once:
seqpipe -m /your/MosaicHunter/directory/MosaicHunter.pipe preparation REFERENCE_DIR=/your/MosaicHunter/directory/Reference TOOLS_DIR=/your/MosaicHunter/directory/Tools
Pre-installed softwares required for the script:
#SeqPipe: 0.4.12
#BEDtools: 2.15.0
#SAMtools: 0.1.18
#FASTX-Toolkit: 0.0.13
#Blat: 34x13
#fastasplitn
Reference data for the script: (Please put them into /your/MosaicHunter/directory/Reference)
#human_g1k_v37.fasta (available at http://soms.nibs.ac.cn:6235/glk/human_g1k_v37.fasta)
#human_g1k_v37.genome (available at http://soms.nibs.ac.cn:6235/glk/human_g1k_v37.genome)
#human_hg19.fasta (available at http://soms.nibs.ac.cn:6235/hg/human_hg19.fasta)
#all_repeats.b37.bed (available at http://soms.nibs.ac.cn:6235/dbsnp/all_repeats.b37.bed)
#PAR.b37.bed (available at http://soms.nibs.ac.cn:6235/dbsnp/PAR.b37.bed)
#dbsnp_137.b37.SNP_AF.tsv (available at http://soms.nibs.ac.cn:6235/dbsnp/dbsnp_137.b37.SNP_AF.tsv)
#observed_in_common.bed (available at http://soms.nibs.ac.cn:6235/dbsnp/observed_in_common.bed)
Tools for the script: (Please put them into /your/MosaicHunter/directory/Tools)
#generate_beta_log10_val_file.r
#count_homopolymer.cpp
#myjoin
#my_join.pl
#PileupFilter.java
#genotyper.pipe
#Yyx_genotype_log10lik_with_precalc_beta.c
#Yyx_real_log10lik_from_baseQ.c
#Yyx_individual_genotyper.c
#LoFreq_call.c
#sam2fa.pl
#blat_best.pipe
#highest-score.pl
#calculate-score-coverage-identity.pl
#intersect_bed12.pipe
#my.grep
#trimBamByBlock.pl
#strand_bias.R
#allele_pos_dist.R
#splitSamByAllele.pl
======Run
To identify pSNM sites from the whole-genome sequencing data, you can run this command:
seqpipe -m /your/MosaicHunter/directory/MosaicHunter.pipe MosaicHunter REFERENCE_DIR=/your/MosaicHunter/directory/Reference TOOLS_DIR=/your/MosaicHunter/directory/Tools TEMP_DIR=/your/temp/directory INPUT_BAM=example.bam [INDEL_CNV_BED=example.bed] PROJECT_NAME=example GENDER=M THREAD_NUM=5
[INPUT_BAM]: the path of your input .bam file, the .bam file should be sorted and indexed
[INDEL_CNV_BED]: the path of a .bed file containing all the CNV and indel-flanking(+/-5bp) regions which will be masked in our pipeline
[PROJECT_NAME]: a string used as the prefix and suffix of the output files's name
[GENDER]: the gender of the subject, "F" for female or "M" for male
[THREAD_NUM]: the maximum number of threads for running the script
Recommended pre-processing of the .bam file:
1) Removing the duplicated, improper-paired, and multi-hit reads
2) Removing the reads with more than three mismatches
3) Processing the reads by GATK's indel realignment and base quality score recalibration
To change the running order and the parameters of the Bayesian genotyper and the error filters, you can edit the scripts of MosaicHunter in /MosaicHunter/MosaicHunter.pipe, according to the user manual of seqpipe.
======Output
The final list of the pSNM candidates could be found at MosaicHunter_[PROJECT_NAME]/[PROJECT_NAME].mosaic.final.tsv
The columns in the final list represent:
1) chromosome
2) position
3) total depth
4) reference nt
5) alternative nt
6) reference depth
7) alternative depth
8) -log10 of posterior probability of ref-hom genotype
9) -log10 of posterior probability of het genotype
10) -log10 of posterior probability of alt-hom genotype
11) -log10 of posterior probability of mosaic genotype
12) population allele fraction in dbSNP 137, -1 for annotated sites without information of allele fraction, -2 for unannotated sites
13) sequence of +/-500bp flanking regions