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Old 11-19-2012, 11:32 AM   #1
pmiguel
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Default Results from 2x250 v2 hardward MiSeq run.

Finally got the hardware upgrade. Our first run completed over the weekend with a cluster density of just over 800 Kclusters/mm^2. It was genomic DNA libs, so it was an easy test for our instrument.

Here are some pngs from SAV:









I presume the error rate and % perfect reads were derived from the ~1% phiX that I spiked in.

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Old 11-20-2012, 05:18 AM   #2
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Could you post also BioAnalyzer electrophoregram, the intensity curves (all Ns, or only C) over the entire run and thumbnails for C at cycles 30, 160, and 240?
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Old 11-20-2012, 05:59 AM   #3
NextGenSeq
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This is similar to what we see.
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Old 11-20-2012, 06:35 AM   #4
pmiguel
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Quote:
Originally Posted by yaximik View Post
Could you post also BioAnalyzer electrophoregram, the intensity curves (all Ns, or only C) over the entire run and thumbnails for C at cycles 30, 160, and 240?


This is a HS Chip with an overlay of the pre-amp (red) and post-amp (8 cycles) in blue. This was one of 4 libraries on the instrument. It is a little goofy in that we started with only about 10 ng of DNA. So we did the normal DNA TruSeq kit, but used adapters from the RNAseq library so would not end up with bad adapter dimer contamination. Also we sonicated shooting for ~700 bp inserts, then did more stringent Ampures than normal (as low as 0.6X) to try to push the size distributions upwards. Also, I did the denaturation at 10 nM, instead of 2 nM to keep the final NaOH concentration lower.

The other 3 libraries were similar.



Weird new v2 intensity profiles.







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Old 11-20-2012, 07:37 AM   #5
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Thanks. This basically confirms what I suspected from my shorter runs. The usable length of fragment is between 1/3 and 1/2 of total length as descending polymerase molecules begin to crowd and interfere with each other in clusters. Or substrate diffusion becomes a limiting factor, or may be both. On shorter fragments this is more visible as cluster numbers begin to drop drastically and intensity curves fall off the cliff. Drop in intensity eventually generates error and aborts the run. In your example bell curves already began to form flattening over their maxima, which are very broad likely due to larger library size heterogeneity. So to take full advantage of 2x250 kit one needs isert size no less than 700 nt, better longer. Perhaps optimal cluster densities with such insert lengths need to be smaller.
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Old 11-20-2012, 07:55 AM   #6
pmiguel
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There are lots of possible factors that contribute to worsening results as the number of cycles increases. Polymerases interfering with each other as they approach the flow cell surface seems a little far fetched.

Also, generally one gets somewhat higher quality with shorter amplicons on Illumina instruments. So that doesn't really fit your hypothesis.

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Old 11-20-2012, 10:29 AM   #7
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Certainly. The hypothesis is what it is - a hypothesis, more data is needed to prove or disprove, but it seems explaining better what I was observing in my runs than ILM techsupport did. I want to prepare longer libraries with narrower fragment distributions and test the hypothesis more.
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Old 11-20-2012, 01:35 PM   #8
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I'm in agreement with Phillip - after all, adapter dimers yield great quality scores...
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