User:Patrick Ongom

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    Patrick Ongom
    Last login:
    Jun 22, 2015 9:16 PM
    Joined:
    Aug 19, 2014
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    2014 genomics
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    Files 2

    FileSizeDateAttached by 
     filtered_Monpu1.r1.fastqMcf FastQC Report.pdf
    r1 quality check using fastqc
    371.69 kB00:49, 10 Sep 2014Patrick OngomActions
     filtered_Monpu1.r2.fastqMcf FastQC Report.pdf
    r2 quality check using fastqc
    371.94 kB00:49, 10 Sep 2014Patrick OngomActions
    Viewing 10 of 10 comments: view all
    In addition to the group work am trying to do more practice, and am getting stuck most of the time.

    I prepared my job file for running fastfqc on the file "Monpu1.genome.rawReads.fastq" as shown below but it cannot run upon submission, always runs for like 5 minutes and dies, the status of my job is always "Q" instead of "R". I cannot figure out what to do next. what have i not done correctly?


    nano practicefastq

    #!/bin/sh-l
    #PBS -N test.fastqc
    #PBS -q scholar
    #PBS -l nodes=1:ppn=16
    #PBS -l walltime=168:00:00
    module load fastqc
    cd $PBS_O_WORKDIR
    pwd

    fastqc Monpu1.genome.rawReads.fastq

    #####submit and check
    qsub practicefastq
    qstat -u oobia

    ##checking the queue

    Job ID Username Queue Jobname SessID NDS TSK Memory Time S Time
    ----------------------- ----------- -------- ---------------- ------ ----- ------ ------ --------- - ---------
    5574818.carter-adm.rca oobia scholar test.fastqc -- 1 1 -- 04:00:00 Q -- edited 02:00, 11 Sep 2014
    Posted 19:47, 3 Sep 2014
    running smoothly
    ######### Cleaning genome sequence using FastqMcf program ################
    ###prepare adapter file and name it "adapters"
    ###then prepare the job file as below:

    nano fastqMcfjob
    #!/bin/sh -l
    #PBS -N FastqMcf_clean
    #PBS -q scholar
    #PBS -l nodes=1:ppn=2
    #PBS -l walltime=04:00:00
    module load ea-utils
    module load java
    cd $RCAC_SCRATCH

    fastq-mcf adapters \
    Monpu1.genome.rawReads.r1.fq \
    Monpu1.genome.rawReads.r2.fq \
    -o filtered_Monpu1.r1.fastqMcf \
    -o filtered_Monpu1.r2.fastqMcf \
    -C 100000 \
    -q 25 \
    -u \
    -x 0.01 \
    -l 100 \
    -S

    ## output line from fastqMcf cleaning###############
    less FastqMcf_clean.o5581363
    Scale used: 2.2
    Filtering Illumina reads on purity field
    Phred: 33
    Threshold used: 251 out of 100000
    Files: 2
    Total reads: 74991761
    Too short after clip: 7518142
    Trimmed 43666104 reads (Monpu1.genome.rawReads.r1.fq) by an average of 14.74 bases on quality < 25
    Trimmed 50837044 reads (Monpu1.genome.rawReads.r2.fq) by an average of 16.92 bases on quality < 25

    grep count results
    -Monpu1.genome.rawReads.r1.fq
    universal adapter forwrd: 196 after cleaning
    universal adapter reverse: 2 after cleaning
    index adapter forward: 50,881 after cleaning
    index adapter reverse: 316 after cleaning

    -Monpu1.genome.rawReads.r2.fq

    universal adapter forwrd: 464 after cleaning
    universal adapter reverse: 48,578 after cleaning
    index adapter forward: 121 after cleaning
    index adapter reverse: 1,587 after cleaning edited 00:58, 10 Sep 2014
    Posted 00:32, 10 Sep 2014
    ############ quality check for reades filterd using fastqMcf ###########
    #!/bin/sh-l
    #PBS -N QMCF_fastq
    #PBS -q scholar
    #PBS -l nodes=1:ppn=1
    #PBS -l walltime=04:00:00
    module load fastqc
    cd $RCAC_SCRATCH
    pwd

    fastqc filtered_Monpu1.r1.fastqMcf filtered_Monpu1.r2.fastqMcf edited 02:01, 11 Sep 2014
    Posted 00:59, 10 Sep 2014
    Aligning cutadapt output to s.cerevisiae (yeast)
    #!/bin/sh -l
    #PBS -N bt2
    #PBS -q scholar
    #PBS -l nodes=1:ppn=1
    #PBS -l walltime=04:00:00

    module use /apps/group/bioinformatics/modules
    module load bowtie2

    cd /scratch/carter/o/oobia

    bowtie2 -x /scratch/carter/o/oobia/BT2/index.bt2 -U /scratch/carter/r/rbuuck/cutout4.fastq \
    -S bowtie2output.sam

    output:

    149983522 reads; of these:
    149983522 (100.00%) were unpaired; of these:
    148903116 (99.28%) aligned 0 times
    187507 (0.13%) aligned exactly 1 time
    892899 (0.60%) aligned >1 times
    0.72% overall alignment rate edited 01:56, 16 Sep 2014
    Posted 22:54, 14 Sep 2014
    File conversions and manipulations:

    1. SAM to BAM;

    #!/bin/sh -l
    #PBS -N SAMS
    #PBS -q scholar
    #PBS -l nodes=1:ppn=1
    #PBS -l walltime=04:00:00
    module use /apps/group/bioinformatics/modules
    module load samtools
    module load bamtools
    cd /scratch/carter/o/oobia/pato
    pwd
    samtools view -bS /scratch/carter/o/oobia/bowtie2output.sam > yeast.bam

    2. Sorting BAM file
    #!/bin/sh -l
    #PBS -N SAMS_sorted
    #PBS -q scholar
    #PBS -l nodes=1:ppn=1
    #PBS -l walltime=04:00:00
    module use /apps/group/bioinformatics/modules
    module load samtools
    module load bamtools
    cd /scratch/carter/o/oobia/pato
    pwd
    samtools sort yeast.bam yeast.bam.sorted

    3. Extracting unmapped reads
    #!/bin/sh -l
    #PBS -N yst_unmapped
    #PBS -q scholar
    #PBS -l nodes=1:ppn=1
    #PBS -l walltime=04:00:00
    module use /apps/group/bioinformatics/modules
    module load samtools
    module load bamtools
    cd /scratch/carter/o/oobia/pato
    pwd
    samtools view -b -f 4 yeast.bam.sorted.bam > yeast_unmapped.bam

    4. Converting BAM to FASTQ
    #!/bin/sh -l
    #PBS -N yst_fqconvrt
    #PBS -q scholar
    #PBS -l nodes=1:ppn=1
    #PBS -l walltime=04:00:00
    module use /apps/group/bioinformatics/modules
    module load samtools
    module load bamtools
    module load BEDTools
    cd /scratch/carter/o/oobia/pato
    pwd
    bedtools bamtofastq -i yeast_unmapped.bam -fq yeast_unmapped.fq edited 23:03, 14 Sep 2014
    Posted 23:02, 14 Sep 2014
    Aligning yeast free sequences to S. Pombe:
    #!/bin/bash
    #PBS -N ystpmbe_align2
    #PBS -q standby
    #PBS -l nodes=1:ppn=2
    #PBS -l walltime=04:00:00
    module load bowtie2
    cd /scratch/carter/o/oobia/pato
    pwd
    bowtie2 -x /scratch/carter/o/oobia/BT2/POMBEindex -U yeast_unmapped.fq -S YST_PMBE2.sam
    Posted 23:06, 14 Sep 2014
    cleaned data:/scratch/carter/o/oobia/bacteria/YPB_unmapped.fq

    Bases and Reads count:
    #!/bin/sh -l

    #PBS -V
    #PBS -N YPBcount
    #PBS -q scholar
    #PBS -l nodes=1:ppn=2
    #PBS -l walltime=4:30:00

    cd /scratch/carter/o/oobia/bacteria

    OP=($(cat YPB_unmapped.fq | grep -v [^ACTGN] | wc --char --line))
    LINES=${OP[0]}
    CHARS=`expr ${OP[1]} - $LINES`
    echo "Reads: $LINES, Bases: $CHARS"

    output:
    Reads: 148903272, Bases: 22235774876 edited 01:12, 16 Sep 2014
    Posted 23:08, 14 Sep 2014
    Job file for final cleaning against bacterial genome:

    #!/bin/bash
    #PBS -N ypbquee
    #PBS -q standby
    #PBS -l nodes=1:ppn=2
    #PBS -l walltime=04:00:00
    module load bowtie2
    cd /scratch/carter/o/oobia/bacteria
    pwd
    bowtie2 -x BACindex -U /scratch/carter/o/oobia/pato/YST_PMBE2_unmapped.fq -S YPB.sam

    file conversion:

    #!/bin/sh -l
    #PBS -N YPBcnvt_que
    #PBS -q scholar
    #PBS -l nodes=1:ppn=2
    #PBS -l walltime=08:00:00

    module use /apps/group/bioinformatics/modules
    module load samtools
    module load bamtools
    module load BEDTools

    cd /scratch/carter/o/oobia/bacteria
    pwd

    samtools view -bS YPB.sam > YPB.bam

    samtools sort YPB.bam YPB.sorted

    samtools view -b -f 4 YPB.sorted.bam > YPB_unmapped.bam

    bedtools bamtofastq -i YPB_unmapped.bam -fq YPB_unmapped.fq
    Posted 01:18, 16 Sep 2014
    Almost no bacterial sequence was detected, overall alignment rate is 0.00%

    148903116 reads; of these:
    148903116 (100.00%) were unpaired; of these:
    148902898 (100.00%) aligned 0 times
    88 (0.00%) aligned exactly 1 time
    130 (0.00%) aligned >1 times
    0.00% overall alignment rate edited 01:32, 16 Sep 2014
    Posted 01:32, 16 Sep 2014
    http://thegenomefactory.blogspot.com/2013/08/paired-end-read-confusion-library.html
    Posted 01:43, 26 Sep 2014
    Viewing 10 of 10 comments: view all
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