Hi All,
I have run into the following problem that I was hoping somebody could help me
with.
I have been trying to demultiplex some Illumina (101bp PE, dual barcoded)
libraries using Picard - e.g.:
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:
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:
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.
Thanks very much in advance,
Kristian
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