Most HiSeq reads demultiplexing as 'Undetermined'; did seq facility screw up?
We sent a library of multiplexed samples prepped with the KAPA HyperCap workflow for sequencing in a single lane of a HiSeq 4000. To our horror, the vast majority (>90%) came back as undetermined.
All of our barcodes are 8 bp, dual index. Here are a few examples from our R1 undetermined FASTQ file:
@E00593:415:HFLKYCCX2:3:1101:4797:1397 1:N:0:NCTGCAAG+NAGGTGAA
@E00593:415:HFLKYCCX2:3:1101:5629:1397 1:N:0:NCGGAATG+NAATGTAG
@E00593:415:HFLKYCCX2:3:1101:5893:1397 1:N:0:NCCAACTG+NTATTGCC
The first base of every barcode has been replaced with an 'N'. Every read is like that. The barcodes are otherwise correct, so theoretically, I could demultiplex the Undetermined files myself if I used a 1-bp mismatch.
Is this normal? I can't work out if every barcode is supposed to begin with 'N' and this is some sort of wildcarding, or if there's been a mistake at the sequencing facility. Similar threads on seqanswers weren't helpful, so this is my first post as a longtime lurker. I'm not very familiar with raw FASTQ data, so I'm sorry if this is a dumb question, but I'm baffled.
Any suggestions would be gratefully received. Thank you!
All of our barcodes are 8 bp, dual index. Here are a few examples from our R1 undetermined FASTQ file:
@E00593:415:HFLKYCCX2:3:1101:4797:1397 1:N:0:NCTGCAAG+NAGGTGAA
@E00593:415:HFLKYCCX2:3:1101:5629:1397 1:N:0:NCGGAATG+NAATGTAG
@E00593:415:HFLKYCCX2:3:1101:5893:1397 1:N:0:NCCAACTG+NTATTGCC
The first base of every barcode has been replaced with an 'N'. Every read is like that. The barcodes are otherwise correct, so theoretically, I could demultiplex the Undetermined files myself if I used a 1-bp mismatch.
Is this normal? I can't work out if every barcode is supposed to begin with 'N' and this is some sort of wildcarding, or if there's been a mistake at the sequencing facility. Similar threads on seqanswers weren't helpful, so this is my first post as a longtime lurker. I'm not very familiar with raw FASTQ data, so I'm sorry if this is a dumb question, but I'm baffled.
Any suggestions would be gratefully received. Thank you!