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  • Demultiplexing problem

    Hi All,

    I have been trying to demultiplex some Illumina (101bp PE, dual barcoded) libraries using Picard - e.g.:

    Code:
    java -Xmx2g -jar /seq/software/picard/1.626/bin/ExtractIlluminaBarcodes.jar BASECALLS_DIR=/analysis/140615/_temp/140615.H9FT9ADXX/Data/Intensities/BaseCalls/ LANE=2 READ_STRUCTURE=101T8B101T BARCODE_FILE=/analysis/140615/barcodeData.2 METRICS_FILE=/analysis/140615/_logs/barcode.metrics.H9FT9ADXX.2.txt MAX_MISMATCHES=0 MINIMUM_BASE_QUALITY=25 NUM_PROCESSORS=1
    I have done this a million times before, but with these new libraries I keep getting the following error messages:

    Code:
    Barcode AGGCAGAA specified more than once in /analysis/140615/barcodeData.2
    Barcode CAGAGAGG specified more than once in /analysis/140615/barcodeData.2
    Barcode CAGAGAGG specified more than once in /analysis/140615/barcodeData.2
    .
    .
    .
    I have never seen this happen before and I have tried everything (I can think of...) to fix the problem. Basically it appears as if Picard isn’t accepting the dual barcodes and just treats them individually - which is strange, since I have demultiplexed many runs that were dual barcoded. If I just to try to demultiplex samples where both barcodes are different, then everything goes through.

    My barcode data files look like this:
    Code:
    barcode_name	library_name	barcode_sequence_1	barcode_sequence_2
    H9FT9ADXX-EM-096_NuGEN	EM-096_NuGEN	AGGCAGAA	TATCCTCT
    H9FT9ADXX-EM-098_NuGEN	EM-098_NuGEN	AGGCAGAA	AGAGTAGA
    H9FT9ADXX-EM-095B_NuGEN	EM-095B_NuGEN	CAGAGAGG	TAGATCGC
    H9FT9ADXX-G3691-1_NuGEN	G3691-1_NuGEN	CAGAGAGG	CTCTCTAT
    H9FT9ADXX-K562B_NuGEN	K562B_NuGEN	CAGAGAGG	CTAAGCCT
    H9FT9ADXX-EM-095_NuGEN	EM-095_NuGEN	CTCTCTAC	CTAAGCCT
    H9FT9ADXX-G3670-1_NuGEN	G3670-1_NuGEN	CTCTCTAC	TAGATCGC
    H9FT9ADXX-G3679-1_NuGEN	G3679-1_NuGEN	CTCTCTAC	TATCCTCT
    H9FT9ADXX-G3680-1_NuGEN	G3680-1_NuGEN	CTCTCTAC	AGAGTAGA
    H9FT9ADXX-G3681-1_NuGEN	G3681-1_NuGEN	CTCTCTAC	GTAAGGAG
    H9FT9ADXX-G3682-1_NuGEN	G3682-1_NuGEN	CTCTCTAC	ACTGCATA
    H9FT9ADXX-G3683-1_NuGEN	G3683-1_NuGEN	CTCTCTAC	AAGGAGTA
    H9FT9ADXX-G3686-1_NuGEN	G3686-1_NuGEN	GGACTCCT	ACTGCATA
    H9FT9ADXX-G3676-1_NuGEN	G3676-1_NuGEN	TAGGCATG	AAGGAGTA
    H9FT9ADXX-G3676-2_NuGEN	G3676-2_NuGEN	TAGGCATG	CTAAGCCT
    H9FT9ADXX-G3677-1_NuGEN	G3677-1_NuGEN	TAGGCATG	GTAAGGAG
    H9FT9ADXX-G3677-2_NuGEN	G3677-2_NuGEN	TAGGCATG	ACTGCATA
    H9FT9ADXX-G3687-1_NuGEN	G3687-1_NuGEN	TAGGCATG	TAGATCGC
    Any idea how to fix this? It's driving me potty.

  • #2
    ..... aaaaand after 3.5h I figured it out - my good old friend 'read structure'! I forgot to change 101T8B101T to 101T8B8B101T

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