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  • Trimming seq data using Trimmomatic confirmation

    Dear 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!

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