scBS-map

• Description: Single-cell Bisulfite Sequencing Data Mapping. scBS-map includes 5 steps:

• Version: 1.0.0

• System requirements

  • Install SAMtools

    • wget samtools-*.tar.bz2
    • tar -xjvf samtools-*.tar.bz2
    • cd samtools-*
    • ./configure --prefix=/samtools_install_path/
    • make
    • make install
    • export PATH=/samtools_install_path/:$PATH

  • Install bowtie2

    • wget bowtie2-*.zip
    • unzip bowtie2-*.zip
    • export PATH=/bowtie2_install_path/:$PATH

  • Install BS-Seeker2

    • wget BSseeker2*.zip
    • unzip BSseeker2*.zip
    • export PATH=/BSseeker2_install_path/:$PATH

• Usage: perl scBS-map.pl [options] [-f <.fastq>] [-g <genome.fa>] [-o <out.bam>]

  -l    Length of trimming bases from the 5' end of the read [default: 10].
  -p    Number of threads. [default: 12].
  -s    Path to samtools eg. /home/user/bin/samtools
        - By default, we try to search samtools in system PATH.
  -a    Path to bs_seeker2-align eg. /home/user/bin/bs_seeker2-align.py
        - By default, we try to search bs_seeker2-ailgn in system PATH.
  -b    Path to bs_seeker2-build eg. /home/user/bin/bs_seeker2-build.py
        - By default, we try to search bs_seeker2-build in system PATH.
  -w    Logical to determine if the genome index needs to be built or not [default: FALSE].
  -n    Length of removing microhomology regions from bam files [default: 10].
  -k    Logical to determine whether to keep temporary files [default: FALSE].
  -f    File name for sequencing reads, .fastq format.
        - a compressed file (.fastq.gz) is also supported.
  -g    Genome file name, fasta format.
  -o    Output file name, bam format.
  -h    Help message.
  

• Example: perl scBS-map.pl -l 9 -p 40 -n 10 -f Sample1.R1.fastq.gz -g hg38.fa -o Sample1.R1.bam

• Output files:

  • .end2end.bam: Output file by end-to-end mode, .bam format.

  • .local.bam: Output file by local mode, .bam format.

  • .unaligned.fq: Unaligned reads, .fq format.

  • .multihits.fq: Multiple-hits reads, .fq format.

  • .out.bam: Final alignment file, .bam format

  • .scBS-map.report: Report for the alignment results.

    --------------------------------------------------
    scBS-map report for test sample
    --------------------------------------------------
    Number of reads: 10000
    Number of bases: 1312217
    Number of reads after quality control: 9852
    Number of bases after quality control: 1219137
    
    Number of mapped reads using the end-to-end mode: 2827
    Number of mapped bases using the end-to-end mode: 356285
    
    Number of mapped reads using the local mode: 1427
    Number of mapped bases using the local mode: 98277
    
    Number of unmapped reads: 4688
    Number of multi-hits reads: 910
    
    Number of mapped reads in total: 4254
    Mappability in total: 42.54%
    
    Number of mapped bases in total: 454562
    Mappability at base level in total: 34.64%
    --------------------------------------------------
    

• Note: scBS-map includes 5 subcommands. You can directly run the scBS-map.pl command for the entire pipeline or select one of the following subcommonds according to your own needs.

• Subcommands:

  1. qcreads:

     • Description: Trim low quality sequences.

     • Usage: perl qcreads.pl [-f <.fastq>] [-l length] [-o output]

       -f FILE               File name for sequencing data, fastq format.
       -l INT                Length of removed bases from the 5' end of the read [default: 10].
       -o OUTFILE            Output file name, .fastq.gz format.
       

     • Example: perl qcreads.pl -f Sample.R1.fastq.gz -l 10 -o Sample.R1.trim.fastq.gz

  2. align-end2end:

     • Description: Perform end-to-end alignment on clean reads.

     • Usage: perl align-end2end.pl [-f input<.fastq>] [-g genome<.fa>] [-p threads] [-u unmappedreads] [-o output]

       -f FILE               File name for clean data, fastq format.
       -g FILE               Genome file name, fasta format.
       -p INT                Number of launching threads [default: 12].
       -u OUTFILE            File name for unmapped reads if needed.
       -o OUTFILE            Output file name, bam format.
       

     • Example: perl align-end2end.pl -f Sample.R1.trim.fastq.gz -g hg38.genome.fa -p 40 -u Sample.R1.unmapped.bam -o Sample.R1.end2end.bam

  3. align-local:

     • Description: Perform local alignment on clean reads.

     • Usage: perl align-local.pl [-f input<.fastq>] [-g genome<.fa>] [-p threads] [-o output]

       -f FILE               File name for clean data, fastq format.
       -g FILE               Genome file name, fasta format.
       -p INT                Number of launching threads [default: 12].
       -o OUTFILE            Output file name, bam format.
       

     • Example: perl align-local.pl -f Sample.R1.clean.fastq.gz -g hg38.genome.fa -p 40 -o Sample.R1.local.bam

  4. qcbam:

     • Description: Remove the low confidence alignments within microhomology regions

     • Usage: perl qcbam.pl [-f <in.bam>] [-n number] [-p threads] [-o <out.bam>]

       -f FILE               File name for local alignment, bam format.
       -n INT                Number of trimming bases [default: 10]
       -p INT                Number of launching threads [default: 12].
       -o OUTFILE            Output file name, bam format.
       

     • Example: perl qcbam.pl -f Sample.R1.local.bam -n 10 -o Sample.R1.local.hc.bam

  5. mergebam:

     • Description: Merge alignments from end-to-end and local mapping if available

     • Usage: perl mergebam.pl [-e <.end2end.bam>] [-l <.local.bam>] [-p threads] [-o output]

       -e FILE               File name of end2end alignment, .bam format.
       -l FILE               File name of local alignment, .bam format.
       -p INT                Number of launching threads [default: 12].
       -o OUTFILE            Output file name, bam format.
       

     • Example: perl mergebam.pl -e Sample.R1.end2end.bam -l Sample.R1.local.hc.bam -p 40 -o Sample.R1.merge.bam

• Contact:

  Peng Wu; wupeng1@ihcams.ac.cn