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Old 04-02-2015, 01:57 AM   #62
Location: UK

Join Date: Jan 2014
Posts: 75

Data in all cases is whole genome sequencing of S.cerevisiae genomic DNA (also containing mitochondiral DNA and an endogenous plasmid element). Prepared using NexteraXT using all standard conditions and sequenced with 2x300bp on MiSeq v3 chemistry. 24 samples multiplexed (and successfully demultiplexed).

The only change was the last run we used less Ampure beads during smaple prep than for first 3 runs (we used the conc recommended in the brochure for 2x250bp, whereas previously we'd used more Ampure beads) to size select larger amplicons.

We think this probably worked because we have less "duplicates" in the SAM file suggesting that fewer paired reads fully overlapped (which occurs when the inserts are shorter than 300bp). The last run was also sequenced at 350+250bp, however, so duplicates will only arise for fragments shorter than 250bp.

The cluster densities for the four runs (in order) are:

Run 1. 1399K/mm2; 90% pass filter; 33.4M clusters, 30.08 pass filter. Created lots of good quality data.
Run 2. 1212K/mm2; 92.3% pass filter; 29.15M clusters; 26.9M pass filter. Created decent data.
Run 3. 999K;mm2; 90.9% pass filter; 24.3M clusters; 22.1M pass filter. Created decent data.
Run 4. 740K/mm2; 77.8% pass filter; 18.4M clusters; 14.45 pass filter. Created data that aligns less well (75% versus 97% for other runs) and has clear quality issues and perfect read issues in PhiX library. It also looks like the bottom flowcell surface was particularly affected.

Could it be that the loading cluster density was too low, impacting clusters passing filter? 77.8% passing is very bad compared to other runs. (740K/mm2 doesn't seem that much below target loading to me though - indeed, the first run 1399, is well above target).

Yet even in those that passed, the data is crappy (in my opinion).

As an example, I attach an image of the PhiX error rate for the bottom surface for the four runs. To me, it looks like run 4 has a massive issue. The top surface is also not as good as the others in read1, but interestingly in read2 actually looks better (second image).

Last edited by M4TTN; 04-02-2015 at 02:09 AM.
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