I have heard a rumour that the long standing MiSeq v3 600 bp kit chemistry problems which led to poor quality read 2 data have recently been solved by Illumina. Anyone know if this is true?
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Yes, the last few 2x300 runs were way better than 1 year ago.
Yes, the last few runs were way better (touch the wood).
If doing amplicons, please use a lot of phiX or shotgun library (up to 20%) (esp. if fist 25 bp's are the same). It would not hurt, and gains in data quality/yield from better dephasing calculation far outweigh the losses from 5%-20% phiX sequence spike in.
If doing cDNA amplicon analysis: make sure you do not use oligoC oligos in your amplicons, because the read quality would go through the floor if there are 10-14C's or G's in a row, and you may also get non-specific amplification of rRNA (rcDNA), reducing usable data yield by 1-2 orders of magnitude.
Also lower the loading density for 500-600bp amplicons, if you need high quality reads for your analysis.
Obviously do not forget, that the longer the amplicon or the read is, the more sensitive it becomes to the DNA sequence content.
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Originally posted by plyguy69 View PostCan anyone confirm this? I need to do some 2x300 sequencing for Ig RNA-seq experiments. Would be awesome if I could use the MiSeq.
@Markiyan - thanks for the advice, I'll pass it on!
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550bp+ full amplicon sequencing platforms...
Originally posted by Brian Bushnell View PostAre there alternatives other than Sanger? Or, what was your backup plan?
2. If you can cope with 2-4% systematic errors - Oxford Nanopore 2D.
3. There used to be Roche's systems - FLX+ and Junior, but the reagents for them are no longer available... :-(
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Not good
It was an amplicon run, but the cluster density was good (~600K) with 20% phiX, so I expected the Q30 to be much better than 64% overall. It was only 54% for Read 2. Both reads only got out to about 150 bp before dropping off dramatically. The investigator decided it was definitely not worth trying to use the 600-cycle kit again!
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Originally posted by microgirl123 View PostNot good
It was an amplicon run, but the cluster density was good (~600K) with 20% phiX, so I expected the Q30 to be much better than 64% overall. It was only 54% for Read 2. Both reads only got out to about 150 bp before dropping off dramatically. The investigator decided it was definitely not worth trying to use the 600-cycle kit again!
Anyone else with recent experience running the 'new' 600 bp kits? We plan to use one this week or next....
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Originally posted by microgirl123 View PostNot good
It was an amplicon run, but the cluster density was good (~600K) with 20% phiX, so I expected the Q30 to be much better than 64% overall. It was only 54% for Read 2. Both reads only got out to about 150 bp before dropping off dramatically. The investigator decided it was definitely not worth trying to use the 600-cycle kit again!
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Adapter dimers?
Originally posted by microgirl123 View PostNot good
It was an amplicon run, but the cluster density was good (~600K) with 20% phiX, so I expected the Q30 to be much better than 64% overall. It was only 54% for Read 2. Both reads only got out to about 150 bp before dropping off dramatically.
Basically significant amount of the clusters on the run contained shorter templates, and once the end of these is reached (140-160 bp), it wrecks RTA dephasing calculations.
since templates in the 150-200 bp cluster very efficiently compared to 500-800 bp ones, it takes only 3-5% of the shorter contaminant to wreck Q scores in the run.
To minimize it avoid repetitive freeze thaw cycles of the Illumina adapters (they can lose T tails and self ligate) and make sure no nucleases contamination is present during the ligation.
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Originally posted by microgirl123 View PostNot good
It was an amplicon run, but the cluster density was good (~600K) with 20% phiX, so I expected the Q30 to be much better than 64% overall. It was only 54% for Read 2. Both reads only got out to about 150 bp before dropping off dramatically. The investigator decided it was definitely not worth trying to use the 600-cycle kit again!
The suggestion that there may be a primer diamer issue is reasonable, but you also may need to up your density a bit.Microbial ecologist, running a sequencing core. I have lots of strong opinions on how to survey communities, pretty sure some are even correct.
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Originally posted by microgirl123 View PostNot good
It was an amplicon run, but the cluster density was good (~600K) with 20% phiX, so I expected the Q30 to be much better than 64% overall. It was only 54% for Read 2. Both reads only got out to about 150 bp before dropping off dramatically. The investigator decided it was definitely not worth trying to use the 600-cycle kit again!Attached Files
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