Hi,
I think I've figured out a problem we have with a recent experiment. We have amplicons of ~400bp with a 5bp index added on to both the 5' and 3' ends (part of the PCR primers). The forward and reverse primers for each PCR have identical indices.
We then pooled equimolar amounts of 50 separate PCRs (each with its own unique 5' and 3' index) and ligated the standard TruSeq adaptors prior to library enrichment as per the TruSeq DNA protocol.
We get good sequence quality, but the paired reads do not match up in terms of index. For example, if read 1 sequences through index GATCA, we would expect read 2 to have the same index (or possibly reverse complement depending on which strand gets sequenced). However, we get a random assortment of read 2 indices.
I think the issue is chimera formation during the second round of PCR when amplifying with the Illumina primers. Because the mixed amplicons are fairly similar except for the indices, they are annealing so that the top strand might be 5'-GATCA index but bottom strand is 5'-TTCTT...
If I'm right, we could solve this using the new PCR-free DNA kits, but I'm uncertain as to DNA input and was wondering if anyone could help? The kit specifies input of 1-2ug of DNA, but we start from the A-tailing step (our amplicons are already at 400bp and blunt-ended so no need to fragment or end repair). My question would be, what sort of input DNA level would Illumina be expecting following the two bead cleanups in the fragmentation and end repair stages? Presumably there will be some loss of template prior to the A-tailing?
Thanks,
Matt
I think I've figured out a problem we have with a recent experiment. We have amplicons of ~400bp with a 5bp index added on to both the 5' and 3' ends (part of the PCR primers). The forward and reverse primers for each PCR have identical indices.
We then pooled equimolar amounts of 50 separate PCRs (each with its own unique 5' and 3' index) and ligated the standard TruSeq adaptors prior to library enrichment as per the TruSeq DNA protocol.
We get good sequence quality, but the paired reads do not match up in terms of index. For example, if read 1 sequences through index GATCA, we would expect read 2 to have the same index (or possibly reverse complement depending on which strand gets sequenced). However, we get a random assortment of read 2 indices.
I think the issue is chimera formation during the second round of PCR when amplifying with the Illumina primers. Because the mixed amplicons are fairly similar except for the indices, they are annealing so that the top strand might be 5'-GATCA index but bottom strand is 5'-TTCTT...
If I'm right, we could solve this using the new PCR-free DNA kits, but I'm uncertain as to DNA input and was wondering if anyone could help? The kit specifies input of 1-2ug of DNA, but we start from the A-tailing step (our amplicons are already at 400bp and blunt-ended so no need to fragment or end repair). My question would be, what sort of input DNA level would Illumina be expecting following the two bead cleanups in the fragmentation and end repair stages? Presumably there will be some loss of template prior to the A-tailing?
Thanks,
Matt
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