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  • Multiple indexes per sample how to trim with Trimmomatic?!

    Hi all,

    I received my HiSeq Illumina RNAseq data and want to run trimmomatic to remove bad quality reads and trim adapters. I have used trimmomatic before but I am currently in doubt if I create the adapter file in a correct way, can someone please correct me if I'm wrong?

    Sequencing happened on Illumina HiSeq4000 using NEBnext kit for the library construction.

    From the sequencing facility I have received the adapter and index sequences (e.g for my first sample):

    Adapter:

    5' Adapter: 5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3'

    3' Adapter(lowercase 6bp bases is Index) 5'-GATCGGAAGAGCACACGTCTGAACTCCAGTCACatcacgATCTCGTATGCCGTCTTCTGCTTG-3

    index:
    CTGGCATA;CGGCTATG

    - I do not understand why I receive two indexes for a single sample? A single sample, needs only one adapter with one specific index? Correct?

    - Trimmomatic needs an adapter file and I will create as follows (with all adapters and indeces in a single file):

    >Prefix_AdapterPE1/1
    AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT
    >Prefix_AdapterPE1/2
    GATCGGAAGAGCACACGTCTGAACTCCAGTCACctggcataATCTCGTATGCCGTCTTCTGCTTG

    With the index in capitals also, but just to show that I will manually add the index sequence. I will do this for all received index sequences, added to a single adapter file and will finally run Trimmomatic.

    Any comments are welcome!

  • #2
    Trimmomatic performs a variety of useful trimming tasks for illumina paired-end and single ended data.The selection of trimming steps and their associated parameters are supplied on the command line.

    The current trimming steps are:

    ILLUMINACLIP: Cut adapter and other illumina-specific sequences from the read.
    SLIDINGWINDOW: Perform a sliding window trimming, cutting once the average quality within the window falls below a threshold.
    LEADING: Cut bases off the start of a read, if below a threshold quality
    TRAILING: Cut bases off the end of a read, if below a threshold quality
    CROP: Cut the read to a specified length
    HEADCROP: Cut the specified number of bases from the start of the read
    MINLEN: Drop the read if it is below a specified length
    TOPHRED33: Convert quality scores to Phred-33
    TOPHRED64: Convert quality scores to Phred-64
    It works with FASTQ (using phred + 33 or phred + 64 quality scores, depending on the Illumina pipeline used), either uncompressed or gzipp'ed FASTQ. Use of gzip format is determined based on the .gz extension.

    For single-ended data, one input and one output file are specified, plus the processing steps. For paired-end data, two input files are specified, and 4 output files, 2 for the 'paired' output where both reads survived the processing, and 2 for corresponding 'unpaired' output where a read survived, but the partner read did not.
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