TruSeq DNA PCR-free Sample Preparation Kit (Part#15036187 Rev.A):extra peak, low qPCR

[snarapor]

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.

[nucacidhunter]

I will not comment on your V3-V4 region sequencing strategy as it has been covered well in this forum and can be find by searching threads. Following suggestions are just to save the work that you have already done.

(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.

The guide has clearly stated that not to size fragments on Bioanalyser because they migrate anomalously due to structure of adapters flanking fragments. The size you see on Bioanalyser trace is not real.

(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.

Firstly, you have measured total DNA concentration with Qubit. QPCR measures a subset of fragments with proper adapters ligated to the ends which are able to form clusters. That is one reason for discrepancy. Secondly, you need correct sizing to calculate your molar concentration by QPCR and guide has proposed how to do it.

*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)?

I would properly measure library concentration according to guide. If concentration is still low, the library can be concentrated by beads or column by eluting in less volume. PCR also can be used to increase concentration and in this case should not have any detrimental effect.

[avo]

Originally Posted by snarapor

(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.

What kit did you use for the qPCR quantification and how much library did you use for the first dilution? You could reduce the volume of the last elution step to 20 µl. If thats not enough you can add a couple of PCR cycles.

[snarapor]

To nucacidhunter, avo
Thank you very much for the prompt reply. I kind of get an idea now.

[snarapor]

2 questions:

(1) In step validate library, we followed TruSeq DNA PCR-free Sample Preparation Kit (Part#15036187 Rev.A). We got 12nM total by qubit vs. 3.4nM total by qPCR. Hence, does this mean the library contain a lot of unligated fragments? If so, could we go back to do from the ligation step -any cons?

Or (2) should we not repeat ligation step but do 2-3 cycles PCR like you recommend?
For PCR to get to 2nM library total. Do we follow the PCR reagents (TruSeq PCR kit (#15026486)) and protocols from page 80, TruSeq DNA Sample Preparation Guide (Part#15026486 Rev.C) followed with magnetic bead cleanup? Could we just use the primer cocktail from the kit to do PCR, followed with minElute for PCR cleanup?


Thank you very much.

Naraporn

[nucacidhunter]

1) In step validate library, we followed TruSeq DNA PCR-free Sample Preparation Kit (Part#15036187 Rev.A). We got 12nM total by qubit vs. 3.4nM total by qPCR. Hence, does this mean the library contain a lot of unligated fragments? If so, could we go back to do from the ligation step -any cons?

Yes. Ligation never is 100% and since your input would be different from Illumina recommendation for the kit, most likely the optimum ratio of adapter to substrate was not achieved. You can repeat ligation and that should be fine, although it is not necessary.

Or (2) should we not repeat ligation step but do 2-3 cycles PCR like you recommend?
For PCR to get to 2nM library total. Do we follow the PCR reagents (TruSeq PCR kit (#15026486)) and protocols from page 80, TruSeq DNA Sample Preparation Guide (Part#15026486 Rev.C) followed with magnetic bead cleanup? Could we just use the primer cocktail from the kit to do PCR, followed with minElute for PCR cleanup?

You can use PCR primer cocktail and master mix from other TruSeq DNA kits. Every PCR cycle will increase your library concentration 1.5-2x each cycle. You can also increase concentration by eluting with less volume. I would recommend using 1x bead for clean-up and not the column. Columns are not good in clearing primer or adapter -dimers which will have negative effect on your library sequencing.