Nextera XT AMPure XP product- no peak with Bioanalyzer?

[ADGM]

Hi guys, I need quick help!

I'm sequencing with an Illumina Miseq for the first time. I'm following the Nextera XT workflow. My samples are S. aureus genomic DNA. I've done up to the lib normalization step but decided to QC my samples before proceeding to loading. I couldn't see any bands with my AMPure products by 2% AGE or Bioanalyzer, although Quantifluor/Qubit shows readings (ranging from 0.3-5 ng/uL). Is it possibly due to the detection limit of the machine (considering how low the starting amount is needed with the Nextera XT workflow), or do you think there is something wrong with my samples?

My initial genomic DNA samples were way high up than the recommended 0.2 ng/ul starting material so I had to dilute it down to get it around that ballpark range. I used Quantifluor for the quantification. It's possible I have thawed the original genomic DNA at least thrice in the dilution process. In the tagmentation step I loaded 5 - 10 uL of the diluted gDNA, depending on how lower than the target 0.2 ng/uL my sample was. I did the PCR clean-up following the 0.6x vol. recommended for >250bp. I've done up to the lib normalization step but decided to QC my samples before proceeding to loading. Since the product post-normalization is noted to be undetectable by Qubit or Bioanalyzer, I tried to QC my AMPure products instead. I tried viewing my PCR clean-up products by 2% AGE but did not see any bands. I quantified them by Quantifluor and got readings ranging from 1 to 6 ng/uL (Qubit gave 20-40% lower readings for select samples that I re-quantified; it's possible I started out with higher amounts than recommended). I ran 12 selected samples by Bioanalyzer and couldn't see a peak, maybe a very subtle one for two samples, which incidentally had the highest concentrations in the lot (2-3 ng/uL by Qubit, or 4-5 ng/uL by Quantifluor), around the 500 bp range. I don't know if the lack of peaks in the Bioanalyzer is due to a limitation in the sensitivity of the machine or if my samples are bad. Should I proceed or re-do the sample prep?

EDIT: My Nextera XT Sample Prep kit is expired by 3 months, but that has not been a problem in previous runs. My Bioanalyzer kit, too, by 1 year, if that could be a factor. I loaded only 1 uL of my sample into the Bioanalyzer chip (the concentration range was 0.3 to 3 ng/uL). All the undetectable samples had concentrations below 1 ng/uL by Qubit (1.6 ng/uL by QF).

[Number6]

Hi ADGM, if you're looking at the material after the bead normalization, the eluted libraries are single-stranded DNA at this point. qPCR or dPCR can be used to quantify your libraries, but they generally don't show up on a bioanalyzer. We often run the pre-normalized libraries to be sure the preps worked. I hope this info helps.

[ADGM]

Sub-optimal Sequencing Run Takeaway

Hi,

I would like to update that the run for my initial set of samples did not push through because of technical problems with the MiSeq machine.

Conveniently, this gave me time to re-do the sample prep from pre-tagmentation quantification (and subsequent dilution), tagmentation and PCR, AMPure XP clean-up (at 0.6x), normalization, etc.:

1. I quantified the PCR clean-up products; yields were still low at 0.3 to 4 ng/uL (quantification done by Promega Quantifluor);

2. The absence of peaks in my initial Bioanalyzer run I chalked up to the low amount loaded (I loaded only 1 uL of clean-up sample after all, and also the chip that I used was not the high sensitivity version). I ran 10 uL of the PCR clean-up sample by 2% AGE instead, and was able to visualize smears, enough to approximate the average bp length of the fragments (I placed it at 500 to 600 bp; so 1 ng/uL = 3 nM conversion).

3. I skipped the bead-based normalization step altogether, as is recommended for <15 nM libraries (see Nextera XT Library Prep: Tips and Troubleshooting Guide);

4. Instead I pooled my PCR clean-up samples to normalize their concentrations to 4 nM (i.e. calculated uL to reach 1.33 ng, following 1 ng/uL = 3 nM conversion), quantified again (because I thought pooled ng/ pooled uL not necessarily equal to 1.33 or 4 nM (?)), diluted the x nM library 1:1 with 0.2 N NaOH then 1:100 with HT buffer to produce a x/200 nM or (x/200)*1000 pM library, quantified again then diluted accordingly with HT1 to reach 12 pM (see Preparing Libraries for Sequencing on the MiSeq).

5. Cluster density was a little below normal range at 744 K; but output was 6.5 GB, with 5 GB or 77% >Q30. I thought run was decent considering my cartridge was pre-thawed and prolonged at 4C for ~2weeks.

Since I re-did the sample prep I ran out of reagents somewhere along the way and had to use really old reagents (>1 year expired) for the ATM (Amplicon Tagment Mix) and NPM (Nextera PCR Mix). But almost all the reagents were expired anyway by at least 3 months.

I sequenced 48 bacterial DNA samples btw.

Conclusion: I think the key for a good run is:

-the normalization (a. if PCR clean-up yield is low, do manual normalization; b. ave. bp length at PCR clean-up should be called correctly so that ng/ul to nM conversion, and therefore dilution, is correct),

-and the library dilution (check concentration post-pooling and dilute accordingly to reach desired pM concentration, while still checking that 1 mM NaOH max final concentration still holds).

-I used expired reagents and a reagent cartridge that was pre-thawed and stored for 2 weeks at 4C but PCR step turned out fine and sequencing run pushed through, respectively, so I guess those factors don't matter as much as the first two.