No human genome has ever been completely sequenced • Hacker News Board

Re: No human genome has ever been completely sequenced

#91

Post by cup-of-tea » Tue, Oct 24, 2017, 11:18 AM UTC

shpx wrote: post #12

jessriedel wrote: post #6

As usual, the journalist spends many paragraphs painting a picture of human conflict before actually getting on to the interesting claim. > The reason for these gaps is that DNA sequencing machines don’t read genomes like humans read books, from the first word to the last. Instead, they first randomly chop up copies of the 23 pairs of chromosomes, which total some 3 billion “letters,” so the machines aren’t overwhelm…

https://youtu.be/fCd6B5HRaZ8 is the best visualization of how the most popular type of DNA sequencer works (that I've found). Imagine you have a string of length 3 billion made by randomly choosing from 4 characters. Like this dna = ''.join(random.choices('atgc', weights=[30.9, 29.4, 19.9, 19.8], k=3_234_830_000)) you get to randomly sample 1 billion[3, page 7] overlapping substrings of length 200[3, page 7] with .1%…

> with .1% of the characters randomly changed

Nope, not randomly. Having a random error profile is the best case. In reality these machines suffer from systematic errors, especially the Illumina ones. That means that they always fail to read through certain pieces of DNA, or make the same error every time.

Re: No human genome has ever been completely sequenced

#92

Post by cup-of-tea » Tue, Oct 24, 2017, 11:21 AM UTC

labster wrote: post #52

Bromskloss wrote: post #46

Earlier quoted context omitted.

Yeah, why does Hacker News not have a real way to indent things? It wouldn't make loading the page any less light-weight.

Seriously. I can't go a week in HN without reading someone's complaint about not being able to read a quote, probably on mobile. If users keep complaining regularly, it's a problem with the software interface, not the users.

Nobody said anything about mobile. Am I missing something?

Re: No human genome has ever been completely sequenced

#93

Post by iskander » Tue, Oct 24, 2017, 12:29 PM UTC

daemonk wrote: post #71

iskander wrote: post #3

It's strange that this article ends as an advertisement for PacBio sequencing (which can ~50k-60k base reads) but makes no mention of Oxford Nanopore (which has gotten megabase reads and keeps improving). Single molecule nanopore sequencing is on track to sequence across the centromeres of human chromosomes in the next few years.

I've personally prep libraries and used nanopore. While there is a lot of promise to the technology, I found it to be too inconsistent. Nanopore claims they can get 10gigs of data, while power users claim they get 2-5gig on average. I consider myself lucky if I get 2gigs of data. The non-random nature of their error profile is also a problem.

Are those experiences on recent chemistries/flow cells? My first library prep gave me ~4 gigabases on a R7.4 flow cell and that was pretty low compared with everyone else in the workshop.

Also, which library prep are you using? I think the ligation kit has historically gotten better yield (not sure if that's true any more).

Re: No human genome has ever been completely sequenced

#94

Post by aroch » Tue, Oct 24, 2017, 2:10 PM UTC

iskander wrote: post #93

daemonk wrote: post #71

Earlier quoted context omitted.

I've personally prep libraries and used nanopore. While there is a lot of promise to the technology, I found it to be too inconsistent. Nanopore claims they can get 10gigs of data, while power users claim they get 2-5gig on average. I consider myself lucky if I get 2gigs of data. The non-random nature of their error profile is also a problem.

Are those experiences on recent chemistries/flow cells? My first library prep gave me ~4 gigabases on a R7.4 flow cell and that was pretty low compared with everyone else in the workshop. Also, which library prep are you using? I think the ligation kit has historically gotten better yield (not sure if that's true any more).

The new rapid kits (R3) have significantly worse performance than the R2 kits it seems.

I'm also extremely put off by how they handle support -- that is via a forum where they do their very best to barely answer anything

Re: No human genome has ever been completely sequenced

#95

Post by daemonk » Thu, Oct 26, 2017, 6:40 PM UTC

iskander wrote: post #93

daemonk wrote: post #71

Earlier quoted context omitted.

I've personally prep libraries and used nanopore. While there is a lot of promise to the technology, I found it to be too inconsistent. Nanopore claims they can get 10gigs of data, while power users claim they get 2-5gig on average. I consider myself lucky if I get 2gigs of data. The non-random nature of their error profile is also a problem.

Are those experiences on recent chemistries/flow cells? My first library prep gave me ~4 gigabases on a R7.4 flow cell and that was pretty low compared with everyone else in the workshop. Also, which library prep are you using? I think the ligation kit has historically gotten better yield (not sure if that's true any more).

Are you working on cell lines or bacteria? Genomic dna? I find most people that tell me they get good data yield are working on samples where dna extraction is relatively straightforward and dna yield is abundant.

I used the flowcell version previous to the current one and got ~2gb of data. I used the ligation kit on genomic extraction of ~10 individuals (crustacean).

Re: No human genome has ever been completely sequenced

#96

Post by daemonk » Thu, Oct 26, 2017, 6:42 PM UTC

aroch wrote: post #94

iskander wrote: post #93

Earlier quoted context omitted.

Are those experiences on recent chemistries/flow cells? My first library prep gave me ~4 gigabases on a R7.4 flow cell and that was pretty low compared with everyone else in the workshop. Also, which library prep are you using? I think the ligation kit has historically gotten better yield (not sure if that's true any more).

The new rapid kits (R3) have significantly worse performance than the R2 kits it seems. I'm also extremely put off by how they handle support -- that is via a forum where they do their very best to barely answer anything

I agree about their communication and support. It does seem like they put all their support behind the few big labs in the UK that are actively using nanopore (free R&D for them I guess). I get a sense of "if you can't get this to work, then you are just a bad scientist" vibe from them.