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Old 12-10-2018, 08:31 PM   #1
African_Skies
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Location: Nairobi

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Smile MiSeq still underclustered at 18 pM

Hello dear people,
We are doing some whole genome sequencing on a plant genome using the MiSeq with v3 reagents for 2x250 paired-end. Library size was measured using a Bioanalyzer and concentration by Qubit.

On the first run, we loaded 15pM library (Nextera), spiked with 1% phiX. We got nice quality (88% >Q30), but a fairly low clustering density (639k/mm2), resulting in 7.3 Gbp of non-index yield.
Surprisingly, the metrics showed 2.3% Ďalignedí reads, though only 1% should be phiX.

Our second MiSeq run used the same library as before. We tried 18pM but went down to 0.5% phiX. The clustering density has increased a bit, but is still on the low side (738k/mm2), quality has not dropped (itís about 91% >Q30), and we got 8.26 Gbp non-index reads.

This makes me wonder whether we are somehow overestimating the effective library concentration due to either underestimating average fragment length (I had quite a broad distribution) or incomplete adapter ligation, causing some fragments to wash off without binding to the flowcell.

In the second run, Ďalignedí reads are still 1.3% despite using 0.5% phiX. Does that mean the effective library concentration that is being sequenced isnít actually 18pM, but more like 6.9 pM (because 0.5% of 18 pM = 1.3% of 6.9pM) and we should go with a significantly higher concentration?

If with v3 reagents we are aiming for a cluster density of 1200-1400, and 12-15 Gbp of yield, are we seriously underclustering and could get much more out of one run by nearly doubling the library concentration? Is there any downside to loading (supposedly) 30pM if more than half of it washes off the flowcell anyway?

Would love to hear what you think.
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Old 12-16-2018, 11:09 PM   #2
African_Skies
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Default Update:

We went ahead with loading 30pM, by using 7.5 uL of 4 nM library and 2.5 ul 0.4N NaOH and then proceeding with the rest of the protocol as if we were handling 20 pM.

The run worked very well, giving us 1152 k/mm2 cluster density, with 87.3% passing the filter. The yield was 11.26 Gbp with 87.5% > Q30, which is barely lower than the quality we had before.

TL;DR:
If your library adapter ligation seems to be incomplete, loading 20 pM on a MiSeq v3 should work fine to compensate.
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Old 01-02-2019, 02:51 PM   #3
ngsmembraneprotein
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Default

Try to measure concentration through qPCR targeting the p7/p5 adapters. This will give you the a good measurement of the concentration that can amplify on the flowcell. You can a kit from NEB for this if you want.
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adapter attachment, cluster density, library concentration, miseq, phix higher than expected

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