[bonsai_spool]

> That's a prevalent misconception even in the scientific community. Sure, each read has 1% incorrect bases (0.01). But each segment of DNA is read many times over. More or less 0.01^(many times) ≈ 0 incorrect bases.

That's true in targeted sequencing, but when you try to sequence a whole genome, this is unlikely.

[optionalsquid]

> That's true in targeted sequencing, but when you try to sequence a whole genome, this is unlikely.

Whole-genome shotgun sequencing is pretty cheap these days.

The person you are replying to doesn't give any specific numbers, but in my experience, you aim for 5-20x average coverage for population level studies, depending on the number of samples and what you are looking for, and 30x or higher for studies where individuals are important.

For context, coverage refers to the (average) number of resulting DNA sequences that cover a given position in the target genome. Though there is of course variation in local coverage, regardless of your average coverage, and that can result in individual base-calls being being more or less reliable

[bonsai_spool]

I’m referring to the experiment done in the OP - the most I’ve read about from an minION flow cell is 8 Gb (and this is from cell line preps with tons of DNA, so the coverage isn’t great).

You need multiple flow cells or a higher capacity flow cell to get anything close to 1X on an unselected genome prep.

Shotgun sequencing isn’t probably what you meant to say - this is all enzymatic or, if it’s sonicated, gets size selected.

[optionalsquid]

What the person you replied to described read like short read sequencing with PCR amplification to me ("each segment of DNA is read many times over"), rather than nanopore sequencing. My reply to you was written based on that (possibly false) assumption.

But if we are talking nanopore sequencing, then yes, you need multiple flowcells. Which is not a problem if you are not a private person attempting to sequence your own genome on the cheap

[bonsai_spool]

There wasn’t enough information to tell (on my 1 minute scan) which nanopore kit was used, but the presence of PCR does not imply short reads.

You can do nanopore PCR/cDNA workflows right up to the largest known mRNAs (13kb).

Edit:

I’m not sure if you’re saying that you can’t do a 5/20/30X genome on nanopore - that’s also not true. It only makes sense in particular research settings, of course.

[zorgster]

PCR is central to short read sequencing... as well as Roche 454, and can also be used in some protocols in Nanopore.

https://pmc.ncbi.nlm.nih.gov/articles/PMC3849550/

You can sequence a human genome on a MinION - but you need to purchase 5 flow cells to reach 11X (if they are used correctly).. https://nanoporetech.com/news/news-human-genome-minion

[bonsai_spool]

> PCR [...] can also be used in some protocols in Nanopore.

Yes... and that is why I said that the presence of PCR does not imply short reads.

> You can sequence a human genome on a MinION - but you need to purchase 5 flow cells to reach 11X (if they are used correctly).. https://nanoporetech.com/news/news-human-genome-minion

THat's why I said that 5/20/30X coverage is possible in the appropriate research setting

[Ovah]

My comment was with NanoPore in mind.

[jakobnissen]

I worked with Nanopore data about four years ago, and I found that that's mostly true, but for some reason at some sites, there was systematic errors where more than half of reads were wrong.

I can't 100% prove it wasn't a legit mutation but our lab did several tests where we sequenced the same sample with both Illumina and Nanopore, and found Nanopore to be less than perfect even with exteme depth. Like, out depth was so high we routinely experienced overflow bugs in the assembly software because it stored the depth in a UInt16.

[bonsai_spool]

What was the DNA source? At the same time (4 years ago) there were issues with specific species - Birds and some metagenome species were the worst if I remember correctly.

[jakobnissen]

Influenza virus