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  • BBDuk quality filtering not producing expected result

    I'm trying to trim/filter low quality reads from paired-end exome-seq data, using BBDuk.

    I used the command:

    ```
    for ea in $files;
    do
    R1="$ea"
    R2=$(echo $R1 | sed "s/R1/R2/")
    /home/shared/programs/bbmap/bbduk.sh -Xmx1g in1=$R1 in2=$R2 \
    out1="$(echo $ea | sed s/.fastq.gz/_trimmed_filtered.fastq.gz/)" \
    out2="$(echo $(echo $ea | sed s/R1/R2/) | sed s/.fastq.gz/_trimmed_filtered.fastq.gz/)" \
    ref=/home/shared/programs/bbmap/resources/adapters.fa \
    t=10 ktrim=r k=23 kmin=11 hdist=1 maq=10 minlen=60 tpe tbo
    done;
    ```

    After running fastqc on the output of this, I'm seeing that R2 files have some reads with low quality scores (see per sequence quality score), and the overrepresented sequence "NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN".

    Looking at these reads in the fastq:
    ```
    @HISEQ:525:HMFYNBCXX:1:1101:1380:2167 2:N:0:CAGATC
    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
    +
    !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
    @HISEQ:525:HMFYNBCXX:1:1101:1276:2219 2:N:0:CAGATC
    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
    +
    !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
    @HISEQ:525:HMFYNBCXX:1:1101:1238:2328 2:N:0:CAGATC
    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
    ```

    Shouldn't these reads have been filtered out?


    Any help here would be much appreciated.
    Last edited by reliscu; 10-13-2021, 09:15 AM.

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