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  • Easiest way to get number of reads per contig?

    Hi all,

    I'm trying to map 2 X 100 PE Illumina reads (transcriptome) back to a small number of contigs. I want to get the number of reads that align to each contig as quickly as possible. Here's the commands I've tried so far but it isn't working.

    Code:
    SCAFFOLD_INPUT_FILE=test_selected_sequences.fas
    S1_FASTQ_INPUT_FILE=D13F4ACXX_s6_1_GSLv2-7_32_SL18114_test.fastq.gz
    S2_FASTQ_INPUT_FILE=D13F4ACXX_s6_2_GSLv2-7_32_SL18114_test.fastq.gz
    REF_MAP_BASENAME=test
    
    BWA=`which bwa`
    SAMTOOLS=`which samtools`
    BCFTOOLS=`which bcftools`
    VCFUTILS=`which vcfutils.pl`
    BT2=`which bowtie2`
    BT_BUILD=`which bowtie2-build`
    TDSTAMP=`date | awk 'OFS="_" {print ($2,$3,$6,$4)}'`
    
    ${BT_BUILD} ${SCAFFOLD_INPUT_FILE} ${REF_MAP_BASENAME}
    ${BT2} -x ${REF_MAP_BASENAME} -1 ${S1_FASTQ_INPUT_FILE} -2 ${S2_FASTQ_INPUT_FILE} --qc-filter --no-unal -S ${REF_MAP_BASENAME}.bowtie2.mapped.sam
    $SAMTOOLS view -bT ${SCAFFOLD_INPUT_FILE} ${REF_MAP_BASENAME}.bowtie2.mapped.sam > ${REF_MAP_BASENAME}.bowtie2.mapped.bam
    $SAMTOOLS sort ${REF_MAP_BASENAME}.bowtie2.mapped.bam ${REF_MAP_BASENAME}.bowtie2.mapped.sorted
    $SAMTOOLS idxstats ${REF_MAP_BASENAME}.bowtie2.mapped.sorted.bam
    Here's the errors I get:
    Code:
    [samopen] SAM header is present: 28 sequences.
    [bam_index_load] fail to load BAM index.
    [bam_idxstats] fail to load the index.
    The problem seems to be once the samtools apps start running. I'm using samtools version 0.1.19-44428cd. Any ideas on what I'm doing wrong? Is there an easier way?

    Thanks!
    Kevin

  • #2
    revise:
    Code:
    ${BT_BUILD} ${SCAFFOLD_INPUT_FILE} ${REF_MAP_BASENAME}
    ${BT2} -x ${REF_MAP_BASENAME} -1 ${S1_FASTQ_INPUT_FILE} -2 ${S2_FASTQ_INPUT_FILE} --qc-filter --no-unal -S ${REF_MAP_BASENAME}.bowtie2.mapped.sam
    $SAMTOOLS view -bT ${SCAFFOLD_INPUT_FILE} ${REF_MAP_BASENAME}.bowtie2.mapped.sam > ${REF_MAP_BASENAME}.bowtie2.mapped.bam
    $SAMTOOLS sort ${REF_MAP_BASENAME}.bowtie2.mapped.bam ${REF_MAP_BASENAME}.bowtie2.mapped.sorted
    $SAMTOOLS idxstats ${REF_MAP_BASENAME}.bowtie2.mapped.sorted.bam
    to

    Code:
    ${BT_BUILD} ${SCAFFOLD_INPUT_FILE} ${REF_MAP_BASENAME}
    ${BT2} -x ${REF_MAP_BASENAME} -1 ${S1_FASTQ_INPUT_FILE} -2 ${S2_FASTQ_INPUT_FILE} --qc-filter --no-unal -S ${REF_MAP_BASENAME}.bowtie2.mapped.sam
    $SAMTOOLS view -bT ${SCAFFOLD_INPUT_FILE} ${REF_MAP_BASENAME}.bowtie2.mapped.sam > ${REF_MAP_BASENAME}.bowtie2.mapped.bam
    $SAMTOOLS sort ${REF_MAP_BASENAME}.bowtie2.mapped.bam ${REF_MAP_BASENAME}.bowtie2.mapped.sorted
    $SAMTOOLS index ${REF_MAP_BASENAME}.bowtie2.mapped.sorted.bam
    $SAMTOOLS idxstats ${REF_MAP_BASENAME}.bowtie2.mapped.sorted.bam
    I added in the call to "samtools index" which is why the samtools idxstats call is failing.
    /* Shawn Driscoll, Gene Expression Laboratory, Pfaff
    Salk Institute for Biological Studies, La Jolla, CA, USA */

    Comment


    • #3
      Thanks! I'm testing it now.

      Comment

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