We have around 80 PCR amplicons, amplified in 3 multiplex pools, that we wish to sequence on a MiSeq. Our products are all between 200-300 bp in size, and we were planning to use the NEBNext® Ultra™ II FS DNA Library Prep Kit for Illumina for the library prep. We were thinking of using 150bp paired end reads. My concern has to do with the initial fragmentation step though.
1. Can we fragment such small products without negatively impacting clustering and read quantity and quality?
2. Can we skip fragmentation entirely, sice the PCR products are around the ideal fragment size anyway?
3. Surely this would be problematic as the DNA fragmentation reagent is also combined with end repair and dA-tailing reagents? Is shearing required for any reason other than generating fragments of the disired size range?
4. If so, should we use a different kit in which these steps are seperate, such as
NEBNext® Ultra™ II DNA Library Prep Kit for Illumina?
I appreciate any insights or comments.
Q: We have around 80 PCR amplicons, amplified in 3 multiplex pools, that we wish to sequence on a MiSeq.
>> 80 samples, is it 80 different PCR reactions? In multiple sequencing context, this means 80 samples for which the library is prepared separately and pooled in the last step for the sequencing. However, I don't unerstand what you mean by "amplified in 3 multiplex pools"??
1. Can we fragment such small products without negatively impacting clustering and read quantity and quality?
>> This kit and fragmentation is primarily done for the genomic DNA, for amplicon sequencing there is no need of fragmentation.
2. Can we skip fragmentation entirely, sice the PCR products are around the ideal fragment size anyway?
>> Yes, as above.
3. Surely this would be problematic as the DNA fragmentation reagent is also combined with end repair and dA-tailing reagents? Is shearing required for any reason other than generating fragments of the disired size range?
>> For multipled amplicon sequencing ready-for-sequencing libraries can be prepared in two step PCR-cleaning-PCR-cleaning program. There is no need for fragmentation, end repair and tailing.
4. If so, should we use a different kit in which these steps are seperate, such as
>> As this kit is primarily used for the whole genome sequencing library prep, and you might also notice that there are many more things required for the library prep which are not provided with the kit. May be you can look for the library prep kit with indexed primers. There are several kits available out there.
* What amplicons are you generating? Maybe including this information in the question might attract better answers.
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