In a previous post I described how our 5% bleach solution for post-run washes had gone bad. Recently I did three MiSeq runs in a row with various amplicons. This was a chance for me to investigate potential carryover contamination between runs, as usually much time passes and multiple washes occur between runs. I was surprised to find a correlation between amplicon read numbers between the first two runs. Carryover levels were around 0.02% , whereas Illumina's Technical Support Note indicates "as low as 0.001% " after carrying out the bleach post-run wash. Between the second and third runs I did the post-run wash twice; this seems to have eliminated any carryover. The current batch of bleach arrived ~8 months ago; this is already too long ago it seems.
Planet Sequencing
Tidbits about DNA (and maybe RNA) sequencing
13 Mar 2022
MiSeq post run washes: an update
3 Dec 2021
ORG.one sequencing success (and some oddities)
ORG.one is an initiative from Oxford Nanopore wherein one can apply for free Nanopore consumables for genome sequencing of critically endangered species. Together with a colleague I applied earlier this year, and we were accepted. The sequencing reagents arrived a few weeks ago - two flow cells and one LSK110 sequencing kit. This is the first time I have used the LSK110 kit (until now I have used LSK109), and it seems to work very well. The yields were high, 34 and 37 Gbases, respectively. These flow cells stayed alive for almost a week (with frequent nuclease flushes). I actually added another 24 hours of sequencing time after 5 days of runtime, and got another ~1.6 Gbase of sequence!
A few oddities during the runs: The translocation speed on the flow cell on our MinION Mk1B was slightly high; starting out just above the green zone. The quality score was also marginally lower than for the other flow cell.
After 4 days, out of nowhere, reads suddenly began going to the "Skipped" folder. A few hours later this behaviour stopped. I have no idea why.
The other flow cell was run simultaneously on our Mk1C. After a nuclease flush, suddenly the pores on the sides of the sensor chip no longer worked, and a large proportion of the channels had changed status to "Saturated". However, multiple manual Mux scans gradually brought them back to life. The same happened on every subsequent nuclease flush.
27 Jul 2021
MiSeq post run washes: beware of expired sodium hypochlorite
Bleach, or sodium hypochlorite (NaOCl) is optionally used during MiSeq post-run washes in order to eliminate run-to-run carryover of library template. I noticed a cloudiness in our 5% sodium hypochlorite. This stock solution was purchased several years ago and stored in the fridge as indicated on the label. The bottle had no expiration date. After some online searching it became obvious that bleach has a (very) limited shelf life, depending on the temperature and concentration. After several years our stock had decomposed to saltwater, and seemed to have some fungal growth! Fortunately our MiSeq seemed unaffected; a cursory check found no indications of run cross-contamination, and a later instrument annual maintenance found the capillaries clear and clean. Nevertheless, the moral of the story is: regularly buy fresh NaOCl for your post-run washes!
20 Jul 2021
Guppy update - "super-accurate" model
Towards the end of May Oxford Nanopore released a new version of the Guppy basecaller. This version includes the Bonito basecaller model, which I previously tested and found that the quality scoring was broken. You can now select among 3 models; fast, HAC, and sup, with sup ("super accurate") the slowest but most accurate. I put our five genomic test datasets through the new version, using the sup model. I am pleased to see that the quality scoring problem from Bonito has been fixed. The sup model shows a small increase in the raw accuracy. This comes at the cost of slower basecalling speeds. In conclusion, another nice upgrade in accuracy. One of these days I must do some assembly benchmarks* to see if this translates into better assemblies! Previous testing by a colleague of mine indicated that this was not always the case.
* I just need to learn how to do assemblies :-)
6 May 2021
2021 phylogenetic tree of Nanopore library kits
It's that time of the year again. It is time for my annual phylogenetic tree of Nanopore library kits. It should be pretty self-explanatory. The devices are:
F - Flongle
M - MinION
G - GridION
P - PromethION
11 Feb 2021
Nanodrop 260/230 ratios decline with time
260/230 ratios as estimated by the Nanodrop can vary a lot among repeat measurements, and decline over time as the drop is left on the pedestal. In contrast, the 260/280 ratio and concentration remains stable over several minutes. I have confirmed this behaviour in multiple tests. I have no idea about the mechanism behind - if it was from evaporation the concentrations should increase. In conclusion - for better accuracy, make replicate measurements, but do so quickly!
28 Jan 2021
Benchmarking Nanopore basecallers: some observations on the Bonito basecaller
We have sequenced several fish genomes on our MinION. Whenever there is a new version of the Guppy basecaller I re-basecall a small dataset from each species and align the raw sequences to previously published, independent references. Using Heng Li's one-liner for sequence identity, I get an estimate of the raw error rate of the sequences.