Hello all. I wonder if anyone has experienced this, and if so how you resolved the problem?
Using our OneTouch V1 with the 200bp V2 DL kit and PGM with 200bp sequencing kit I am experiencing a large peak ~45bp from a 16S V3 library that should be closer to 200bp after barcode and adaptor trimming. Both instruments are working correctly. Our Qiaxcel Advanced shows the short fragments are not "real". Pre-enrichment figures are good (20%). I spiked in the 200bp control library one the same chip and got a mean read length of 216bp. The only cause that remains on the table is the amplicons forming hairpins or some such, which makes no sense as a) lots of other people use V3, b) another group has achieved good results with the exact same protocols, and c) I would expect my library prep to fail were the amplicons forming hairpins. I am using fusion primers with a 10bp barcode and have confirmed that the primers do not form hairpins. Any insight would be much appreciated as this is driving me crazy!
Using our OneTouch V1 with the 200bp V2 DL kit and PGM with 200bp sequencing kit I am experiencing a large peak ~45bp from a 16S V3 library that should be closer to 200bp after barcode and adaptor trimming. Both instruments are working correctly. Our Qiaxcel Advanced shows the short fragments are not "real". Pre-enrichment figures are good (20%). I spiked in the 200bp control library one the same chip and got a mean read length of 216bp. The only cause that remains on the table is the amplicons forming hairpins or some such, which makes no sense as a) lots of other people use V3, b) another group has achieved good results with the exact same protocols, and c) I would expect my library prep to fail were the amplicons forming hairpins. I am using fusion primers with a 10bp barcode and have confirmed that the primers do not form hairpins. Any insight would be much appreciated as this is driving me crazy!
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