Long Read Sequencing- why did we choose 50X coverage...

Our new LONG READ microbial WGS service offers an impressive 50X target coverage of your organism's genome. But what is coverage, and why 50X??

Coverage is an indication of - on average - how many times a given nucleotide within a genome has been sequenced. For example, 50X coverage of a 5 Megabase E. coli genome means we would have sequenced 250 million bases.?

But why does that matter? Try and say this out loud as quickly as possible

“Irish Wristwatch”.

Difficult, right??

But if you say it again and again, and compare each iteration, you can work out which parts are wrong and right. Sometimes you need to repeat things more to really understand where you’re going wrong.?

We can find and fix small errors arising from lower raw read accuracy by creating redundancy in the amount of times we sequence a stretch of DNA. Do it enough times, and you can surpass the inherent basal error rate of your sequencing device.?

How much coverage you need is a tricky question, and it’ll be different for each organism. Ryan Wick recently made an excellent blog post comparing different coverage levels: https://rrwick.github.io/2023/11/06/accuracy-vs-depth-update.html.?

Sometimes more coverage is better, and sometimes more coverage is worse - but 50X was shown to be the best option for a variety of organisms and GC skews in our testing.

But don't just take our word for it, here's a protocol from Oxford Nanopore Technologies themselves; https://community.nanoporetech.com/docs/prepare/library_prep_protocols/no-miss-isolate-sequencing-rapid-barcoding-v14/v/iso_9205_v114_revb_20mar2024/overview-of-the-protocol?devices=minion.

You can also check out this paper published in The Journal of Microbial Genomics by the Microbiology Society ; https://www.microbiologyresearch.org/content/journal/mgen/10.1099/mgen.0.001246.

If you would like to try our LONG READ service you can get a quote here: https://microbesng.com/request-quote/.

Of course long read only assemblies aren’t infallible, and there are still improvements to be made on the sequencing and informatics side. If you need reference quality assemblies, instead of chucking more and more long read coverage at the problem, use our hybrid service!

Hybrid combines structurally informative long reads with the high-raw read accuracy of Illumina short reads. This polishes out errors in the assembly and leaves you with a highly accurate genome.?