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Hi All,
we have a strange problem - fortunately only with a single truseq library. Two independent Hiseq runs resulted in only about 15% usable (demultiplexable) reads from genomic DNA samples.
A whopping 35% of the 6bp barcodes were read as "GGGGGG". These and other very G-rich sequences are dominating the barcode reads. These sequences do not match the actually used barcodes (which were 12 balanced truseq barcode sequences - no bias towards certain bases per position and balanced with regard to the samples).
The quality scores for the barcode reads are poor, as you can imagine, however the forward and reverse reads are fine (and they contain the expected sequences; there is no sign of any external contamination).
The libraries were generated PCR free and we actually only have barcoded Illumina adpters in the lab (truseq style). This is the first time we ran into this. Nevertheless it would be great to know how to prevent reoccurrences.
Did anybody ever come across a similar problem or has a potential explanation?
Thanks a lot in advance!
(We had the libraries sequenced at a service).
we have a strange problem - fortunately only with a single truseq library. Two independent Hiseq runs resulted in only about 15% usable (demultiplexable) reads from genomic DNA samples.
A whopping 35% of the 6bp barcodes were read as "GGGGGG". These and other very G-rich sequences are dominating the barcode reads. These sequences do not match the actually used barcodes (which were 12 balanced truseq barcode sequences - no bias towards certain bases per position and balanced with regard to the samples).
The quality scores for the barcode reads are poor, as you can imagine, however the forward and reverse reads are fine (and they contain the expected sequences; there is no sign of any external contamination).
The libraries were generated PCR free and we actually only have barcoded Illumina adpters in the lab (truseq style). This is the first time we ran into this. Nevertheless it would be great to know how to prevent reoccurrences.
Did anybody ever come across a similar problem or has a potential explanation?
Thanks a lot in advance!
(We had the libraries sequenced at a service).
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