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ChimeraMiner

A pipeline for searching the chimeric sequences in the NGS sequence data.

Reference Genome Preparation

In my server, my reference genome fold is:

/home/luna/Desktop/database/homo_bwa

In this fold, we have 26 fasta files (chr{1..22, X, Y, MT}.fa hsa.fa), each fasta file have indexed with bwa index.

In additional, hsa.fa include all sequence from all chromosome (chr{1..22, X, Y, MT}).

Index for each fasta file:

for chr in 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y MT
do
	bwa index -a bwtsw chr$chr.fa &>> bwa-index.log
	samtools faidx chr$chr.fa &>> samtool-index.log
done
bwa index -a bwtsw hsa.fa &>> bwa-index.log
samtools faidx hsa.fa &>> samtool-index.log

Perl Modules Preparation

In this pipeline, we need some Perl Modules, we should install these modules before run it.

  1. Getopt::Long
  2. Cwd qw(abs_path)
  3. File::Basename
  4. File::Spec

I recommand to use "cpanm" to install Perl Modules.

Test

The Test folder contains an example. It turns out that all the scripts are running. You can check out how to use the pipeline. In this folder, just run workstep.sh first, this shell will generate bam file and chimera's files. When all works in workstep.sh finished, then run filterstep.sh, this shell will deal with the chimera's files and count.

First, align reads to reference, use bwa mem, generate a list contains "SampleID BAMFile";

ref=/home/luna/Desktop/database/homo_bwa/hsa.fa
bwa mem -t 20 -k 30 -R '@RG\tID:Test\tLB:Test\tSM:Test\tPL:ILLUMINA' $ref test_R1.fq.gz test_R2.fq.gz | awk '$6 !~ /H/' | samtools view -Sb -t ${ref}.fai -o $dir/Test/Test.dh.bam -
echo -e "Test\t$dir/Test/Test.dh.bam" > bam.lst

Generate_Shell_Finder.pl

Generate a shelle script for run the ChimeraMiner.

perl Generate_Shell_Finder.pl -i bam.lst -o runFinder.Test.sh -L 20 -r $ref

Insertion.SRExtract.ReConFastq.pl

for first alignment file:
	Step 1:
			seleted first-type chimeric reads	(++ or --).
	Step 2:
			Extract the sequences which are soft-aligned with xxSxxM , xxMxxS or xxSxxMxxS in alignment format from BWA-aligned SAM file.
			        Any Soft-aligned fragment mustn't shorter than log4(Chromosome_Length) nt.
	Step 3:
			Reconstruct FASTQ file from all qualified soft-aligned reads. 
			Qualified reads would be cut into two or three part according to the soft-aligned format.
			All reads would be separated into different chromosomes according to the primary alignment results by BWA.
			Cut reads generated in here would be aligned to Hg19 reference by using BWA-sampe mode.

SearchOverlapSEchimera.pl

Search the overlap of the mapped two segment. This only for single-end chimeric sequences.

dir=/home/luna/Desktop/Tools/git/ChimeraMiner
perl Insertion.SRExtract.ReConFastq.pl -i $dir/Test/Test.dh.bam -m Test -d $dir/Test -r $ref

A1_Extract.Chimeras.pl A2_TransFormat.pl A3_GetInfo.Chimeras.PRE.pl

These 3 scripts provide filtering, classification, and statistics for single-end chimeric sequences.

perl $dir/A1_Extract.Chimeras.pl -d $dir/Test/chimeras -n Test -L 20
perl $dir/A2_TransFormat.pl -i $dir/Test/chimeras/chimeras_analysis/normal.txt -o $dir/Test/chimeras/chimeras_analysis/normal.TransFormat
perl $dir/A3_GetInfo.Chimeras.PRE.pl -i $dir/Test/chimeras/chimeras_analysis/normal.TransFormat -d $dir/Test/chimeras/chimeras_analysis/Test.direct -v $dir/Test/chimeras/chimeras_analysis/Test.inverted  > Test.stat

the usage of bwa

see the details in the page.

Contact:

We will be pleased to address any question or concern you may have with the ChimeraMiner: [email protected]

Citing ChimeraMiner

If you use ChimeraMiner in your work, please cite:

Lu, N.; Li, J.; Bi, C.; Guo, J.; Tao, Y.; Luan, K.; Tu, J.; Lu, Z. ChimeraMiner: An Improved Chimeric Read Detection Pipeline and Its Application in Single Cell Sequencing. Int. J. Mol. Sci. 2019, 20, 1953.