So how do you actually isolate one chromosome to sequence it?
Their github has lots of information about what they do:
Researchers generate complete human X chromosome sequence
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Re: Researchers generate complete human X chromosome sequence
#12The press release is better than the link: https://www.genome.gov/news/news-release/NHGRI-researchers-g...
Ok, changed from https://phys.org/news/2020-07-scientists-human-chromosome.ht.... Thanks!
Re: Researchers generate complete human X chromosome sequence
#13In a simple English, could someone explain why is this good and what does it all mean?
From the article: "Repetitive DNA sequences are common throughout the genome and have always posed a challenge for sequencing because most technologies produce relatively short "reads" of the sequence, which then have to be pieced together like a jigsaw puzzle to assemble the genome. Repetitive sequences yield lots of short reads that look almost identical, like a large expanse of blue sky in a puzzle, with no clues…
> Repetitive sequences yield lots of short reads that look almost identical, like a large expanse of blue sky in a puzzle, with no clues to how the pieces fit together or how many repeats there are
Ah, https://en.wikipedia.org/wiki/Clock_recovery !
Too bad DNA isn't a run-length limited code. (Wouldn't that be something.)
Re: Researchers generate complete human X chromosome sequence
#14[deleted]
Re: Researchers generate complete human X chromosome sequence
#15The article doesn’t mention but now I’m curious what kind of read length and error rate they are achieving. This could have huge impacts across all sequencing.
Re: Researchers generate complete human X chromosome sequence
#16So how do you actually isolate one chromosome to sequence it?
Step 1: the researchers use a diploid cell line where the entire diploid X chromosome is homozygous: both copies of the X chromosome in this cell line are identical. This is part of why the researchers chose to look at the X chromosome.
> To circumvent the complexity of assembling both haplotypes of a diploid genome, we selected the effectively haploid CHM13hTERT cell line for sequencing (abbr. CHM13)
Incidentally, they do capture the other chromosomes in this process:
> Several chromosomes were captured in two contigs, broken only at the centromere (Fig 1a).
> https://www.biorxiv.org/content/10.1101/735928v3.full.pdf
Step 2: Follow a procedure for DNA prep that results in long stretches of DNA (though not an entire chromosome-length) and amplify (make multiple copies of) the mixture, per this reference:
> https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5889714/
Step 3: Run the mixture through a nanopore sequencer (essentially a hole a few nanometres across), reading the change in current in response to the different bases, including methylated bases.
Step 4: Repeat this many many times to get multiple reads of each region of the data:
> In total, we sequenced 98 MinION flow cells for a total of 155 Gb (50× coverage, 1.6 Gb/flow cell, SNote 2). Half of all sequenced bases were contained in reads of 70 kb or longer (78 Gb, 25× genome coverage) and the longest validated read was 1.04 Mb.
Step 5: Overlay the data from the long measurements
> Once we had collected sufficient sequencing coverage for de novo assembly, we combined 39× of the ultra-long reads with 70× coverage of previously generated PacBio data 18 and assembled the CHM13 genome using Canu 19. This initial assembly totaled 2.90 Gbp with half of the genome contained in contiguous sequences (contigs) of length 75 Mbp or greater (NG50), which exceeds the continuity of the reference genome GRCh38 (75 vs. 56 Mbp NG50).
> The read was placed in the location of the assembly having the most unique markers in common with the read. Alignments were further filtered to exclude short and low identity alignments. This process was repeated after each polishing round, with new unique markers and alignments recomputed after each round.
Step 6: Check up the data against the reference genome:
> The corrected contigs were then ordered and oriented relative to one another using the optical map and assigned to chromosomes using the human reference genome.
> The final assembly consists of 2.94 Gbp in 590 contigs with a contig NG50 of 72 Mbp. We estimate the median consensus accuracy of this assembly to be >99.99%.
Essentially, this work closes up difficult-to-read gaps in the reference genome ( https://en.wikipedia.org/wiki/Reference_genome#Human_referen... )
Re: Researchers generate complete human X chromosome sequence
#17In a simple English, could someone explain why is this good and what does it all mean?
From the article: "Repetitive DNA sequences are common throughout the genome and have always posed a challenge for sequencing because most technologies produce relatively short "reads" of the sequence, which then have to be pieced together like a jigsaw puzzle to assemble the genome. Repetitive sequences yield lots of short reads that look almost identical, like a large expanse of blue sky in a puzzle, with no clues…
So 20 years ago when we "sequenced the human genome", we actually didn't? If you'd asked me whether or not we had a complete sequence of an X chromosome before I saw this I would have said, "Of course we have one, for over 20 years".
Re: Researchers generate complete human X chromosome sequence
#18go banana slugs!
Re: Researchers generate complete human X chromosome sequence
#19So how do you actually isolate one chromosome to sequence it?
Step 1: the researchers use a diploid cell line where the entire diploid X chromosome is homozygous: both copies of the X chromosome in this cell line are identical. This is part of why the researchers chose to look at the X chromosome. > To circumvent the complexity of assembling both haplotypes of a diploid genome, we selected the effectively haploid CHM13hTERT cell line for sequencing (abbr. CHM13) Incidentally, t…
Great write up! Thanks!
Regarding step 1 how can any human have an entire homozygous X chromosome?
Also/rather why not just use a male with one X chromosome?
Re: Researchers generate complete human X chromosome sequence
#20The article doesn’t mention but now I’m curious what kind of read length and error rate they are achieving. This could have huge impacts across all sequencing.
It looks like they're using Oxford nanopore and PacBio sequencing technologies for the long reads. These are two up-and-coming sequencing technologies focused on extremely long reads. My understanding of both is that their error rates on individual base pairs are too high to reliably determine the actual sequence on their own (something like 15% error rates). Typically the long reads from these technologies are used as a "scaffold" to resolve the large-scale structure of a DNA sequence, while another sequencing technology, usually Illumina, is used to resolve the actual sequence. (Illumina produces short reads, but it produces a lot of them, and the error rate is much lower, about 1%-5%.) In addition, since PacBio and Oxford Nanopore are very different technologies, I'm guessing that they probably have different "error profiles", so they probably partially cover for each others' deficiencies when you use both of them at the same time.
Note: Don't take any of the specific numbers above as gospel. These technologies develop extremely quickly, so it's quite likely that my knowledge of typical error rates is out of date.
In any case, here's the relevant quote from the original link (to phys.org), before it was changed to the less technical press release, which doesn't mention any specific technologies used:
"The new project built on that effort, combining nanopore sequencing with other sequencing technologies from PacBio and Illumina, and optical maps from BioNano Genomics. Using these technologies, the team produced a whole-genome assembly that exceeds all prior human genome assemblies in terms of continuity, completeness, and accuracy, even surpassing the current human reference genome by some metrics."