Demultiplexing problem

[kga1978]

Hi All,

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.

[kga1978]

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