Subject: TruSeq DNA PCR-free Sample Preparation Kit (Part#15036187 Rev.A): validate library showed extra peak by Bioanalyzer and low qPCR concentration
Hi,
I have a question regarding the TruSeq DNA PCR-free Sample Preparation Kit. We did 16S rRNA V3-V4 amplicon sequencing. The initial consisted of 23 samples of 16S rRNA V3-V4 amplicons of sizes 440 bp, each amplicon was appended 8-nucleotides on the right and left ends. The 8-nucleotides were TCTCTGTG, TCTACTCG, TAGTAGCG, AGACGACG, ACTCGTAG, ACATCGAG, ACGCTATC, TACTACGC, AGCAGAGC, TCAGCTAC, AGAGCGAC, ATGCTCAC, TAGCACAC, TGTACGTG, TAGCTCTG, ACAGATCG and TCACAGCG, for examples. We qubit-measured the dsDNA concentration to be 47.2 ng/ul for 50ul and the size to be 440+8*2 = 456 bp.
Subsequently, we followed the TruSeq DNA PCR-free Sample Preparation Kit and Guide (Part#15036187 Rev.A), starting from page 55 "clean up fragmented DNA" to page 114 "validate library." Then we found 2 problems:
(1) our final product should be about 576 bp but we saw in HS chip Bioanalyzer a peak of 662bp and an extra peak at 1,133 bp. The latter is the double size peak.
(2) the protocol for the next step requires 2nM but the concentration validated by qPCR was only 0.2 nM per peak. The dsDNA concentration measured by Qubit was 12.14 nM total.
*We would like to continue the Illumina protocol "Preparing Libraries for Sequencing on the MiSeq (Part# 15039740 Rev.D)." Due to the extra-peak and the low-concentration by qPCR, could anyone please give me an advice for how to do next?
**Or could we cut both 662 and 1,133 bp bands and PCR to get 2 nM DNA (using reagents from the final step of the TruSeq PCR kit)?
Additional question:
Our initial sample is prepared from
metagenomic DNA -> 16S rRNA PCR amplicons with 8-nt on each ends -> agarose gel electrophoresis -> 456 bp gel extracted DNA -> pool 23 different amplicon libraries each with unique 8-nt barcodes -> make them to final concentration of 2ug in 50ul using HiYield Gel/PCR DNA Fragment Extraction Kit (Invitrogen)
#The question is that since our initial is PCR clean-up product by Invitrogen, can we start the TruSeq DNA PCR-free Sample Preparation Kit and Guide (Part#15036187 Rev.A) from page 57 "Perform End Repair and Size Selection" and omit page 55 "clean up fragmented DNA"?
Thank you very much.
Hi,
I have a question regarding the TruSeq DNA PCR-free Sample Preparation Kit. We did 16S rRNA V3-V4 amplicon sequencing. The initial consisted of 23 samples of 16S rRNA V3-V4 amplicons of sizes 440 bp, each amplicon was appended 8-nucleotides on the right and left ends. The 8-nucleotides were TCTCTGTG, TCTACTCG, TAGTAGCG, AGACGACG, ACTCGTAG, ACATCGAG, ACGCTATC, TACTACGC, AGCAGAGC, TCAGCTAC, AGAGCGAC, ATGCTCAC, TAGCACAC, TGTACGTG, TAGCTCTG, ACAGATCG and TCACAGCG, for examples. We qubit-measured the dsDNA concentration to be 47.2 ng/ul for 50ul and the size to be 440+8*2 = 456 bp.
Subsequently, we followed the TruSeq DNA PCR-free Sample Preparation Kit and Guide (Part#15036187 Rev.A), starting from page 55 "clean up fragmented DNA" to page 114 "validate library." Then we found 2 problems:
(1) our final product should be about 576 bp but we saw in HS chip Bioanalyzer a peak of 662bp and an extra peak at 1,133 bp. The latter is the double size peak.
(2) the protocol for the next step requires 2nM but the concentration validated by qPCR was only 0.2 nM per peak. The dsDNA concentration measured by Qubit was 12.14 nM total.
*We would like to continue the Illumina protocol "Preparing Libraries for Sequencing on the MiSeq (Part# 15039740 Rev.D)." Due to the extra-peak and the low-concentration by qPCR, could anyone please give me an advice for how to do next?
**Or could we cut both 662 and 1,133 bp bands and PCR to get 2 nM DNA (using reagents from the final step of the TruSeq PCR kit)?
Additional question:
Our initial sample is prepared from
metagenomic DNA -> 16S rRNA PCR amplicons with 8-nt on each ends -> agarose gel electrophoresis -> 456 bp gel extracted DNA -> pool 23 different amplicon libraries each with unique 8-nt barcodes -> make them to final concentration of 2ug in 50ul using HiYield Gel/PCR DNA Fragment Extraction Kit (Invitrogen)
#The question is that since our initial is PCR clean-up product by Invitrogen, can we start the TruSeq DNA PCR-free Sample Preparation Kit and Guide (Part#15036187 Rev.A) from page 57 "Perform End Repair and Size Selection" and omit page 55 "clean up fragmented DNA"?
Thank you very much.
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