Whole Genome Sequencing
sequencing.com
sequencing.com
>> Do you sell or share my data with anyone?
> No, we do not sell or share your data, including your DNA data, with anyone.
However, in their privacy policy:
> Your Personal Information may be shared in the following ways:
> With our service providers, allowing them to provide their services to us.
edit:
More clearly stated here:
https://sequencing.com/ordering-dna-test-kit-and-genome-sequ...
> When you purchase the Ultimate DNA Test or Ultimate Genome Sequencing test, your test will be processed by our Laboratory Partners. We will provide our Laboratory Partner with only your basic contact information so that it can send you a DNA collection kit; we will not share your billing information with our Laboratory Partner but will pass on your payments to them.
That does not mean they are sharing your genetic data, maybe just address phone number etc.
I had the WORST experience with Nebula Genomics which ended up with them refunding my money after waiting 6 months.
I hope this is not another one of those companies. I still have some blank spots I am trying to understand from my 23andMe results and this would be great.
(1) Nebula is our laboratory partner, and they sequence with high-throughput MGI DNBSEQ-T7 DNA sequencing machines.
(2) They are our laboratory provider, but the product is not the same! The whole genome sequencing product sold by Nebula on their site is different from the Ultimate Genome Sequencing service we offer in some key ways. One big one is that our service includes more than $200 in DNA analysis apps and reports (such as the Healthcare Pro report designed for healthcare providers and the Rare Disease Screen that analyzes thousands of rare diseases).
The biggest difference, however, is in the processing of the raw genetic data into data files that you can then use to get health insights. We have enhanced processing for these kits that is able to create insights not available from Nebula directly, including enhanced raw data processing through two special bioinformatics pipelines that provide comprehensive data and analysis of structural variations, copy number variations, and mitochondrial heteroplasmy. Both Telomere Length and HLA Typing are coming soon and will be retroactively added to all past purchases of our Ultimate Genome Sequencing service.
This is all fed into our One Genome technology, which allows you to do analysis in ways that you can't do anywhere else. This technology takes all of your genetic data and turns it into an enhanced virtual genome. This advantage of this is that you can run analysis on everything all at once, where in some other cases you might have separate files that can't be analyzed simultaneously.
The long and short of it is that it takes a lot of processing to turn raw genetic data into usable data, and then to analyze that data. That piece is where we come in, and are at the forefront of the field.
> At its discretion, Nebula may use third-party laboratories to perform DNA extraction, sequencing, and data analysis.
(https://nebulagenomics.zendesk.com/hc/en-us/articles/3600243...)
I would guess the samples are eventually sequenced by BGI although Nebula FAQ (https://nebula.org/faqs/) says they sequence in EU.
Thank you so much for revealing this to everyone.
Anyway, an US lab won't care about it.
The US lab is marketing to EU customers. They will care about GDPR (heck, they even claim to be compliant on their website).
I heard good things about Sequencing.com, but then again also Nebula Genomics when I bought their service end of 2021. I ended up asking a refund due to the painfully slow processes and communication last week. Dante Labs is a previous pioneer but has also fallen from grace with long delays and undelivered data. Caveat Emptor.
Still, ONT users would need to understand how to convert raw reads into useable basecalls at loci of clinical interest
Last I looked into it, it was a minimal set of extra equipment -- votexer, centrifuge, etc... nothing unusual for a bio lab. And even most of that could be handled with a good snap of the wrist, if you weren't trying to be super precise.
When your first run fails, are you willing to pay again as much for the kits to run it again (these machines are very much following the cheap printer/expensive ink model IMO)?
Once you have some data, do you know how you will BLAST it against annotated genomes to figure out if you have mutation X? How do you interpret e-scores, etc?
For the OP company, when they say "download the data" do they mean the raw reads, or assemblies? Make sure this is spelled out (it likely is, I haven't looked lately). Do the downloads/service provide adequate metadata on the data generation process so that you can tease out errors in reads from reality (real single-nucleotide mutations), etc.?
All fun stuff, but only for a very serious hobbyist, so to speak.
On the products page it says they provide FASTQs, aligned BAM and VCF. Which is exactly what one would expect. They're almost certainly just running the DRAGEN pipeline (or something similar) and giving you the output it creates.
You aren't trying to sequence the human genome from nothing, but instead have a reference genome to work with. You take each of the reads you get from the sequencer and find where it best matches up with the reference genome.
I think the wet portion is a far bigger challenge for the typical computer programmer hobbyist.
(Also I don't think $1,000 will get you, in addition to the sequencer, a flow cell sufficient to gather enough reads to tell the difference between sequencing errors and mutations?)
Alignment. You could easily automate alignment of raw sequencing reads to their position in the reference genome. From there, even variant calling (finding your individual changes from the reference) is pretty easy as well.
Assembly is a bigger challenge that takes the raw reads and puts them together into a larger (graph) structure. This is usually de-novo, but could be able to be done with a reference as well. But this is not really something that is routine or possible with 30X sequence coverage to the degree one would expect.
The wet-lab portion isn't terrible and anyone with skills making anything (soldering, for example), should be able to do it -- once you figured out some of the jargon.
The bigger challenge someone not trained in this would have is interpreting the variations that you'd find. This can be quite difficult to figure out if an A>C at a particular position is meaningful or not. Again, much of these can be filtered using existing tools, but even then, it can be quite difficult to interpret results.
Whoops; fixed!
> This can be quite difficult to figure out if an A>C at a particular position is meaningful or not.
What about https://promethease.com ?
Note: I do this work for cancer genomes (WGS, tumor and germline). Promethease is not something that I use for annotations, but my workflows are significantly more complex.
The biggest thing I’d look out for is mindset. Biology is very different from comp sci. CS is very deterministic. You change something here, you get a result there. Biology, on the other hand, is very messy. There are compensation mechanisms on top of compensation mechanisms, and everything favors life. If you see a deleterious variant, that still doesn’t mean you’ll see a phenotype (effect on you). It’s quite a different world and can be very scary if you are looking at your own data.
And to complicate things further, one variant in a gene could do nothing if the other copy is still good. Even if you have two bad variants in a single gene, they might be on the same allele (from the same parent). In which case, you still have one good copy! It’s like how Iceman got hit twice in the same engine at the end of the original Top Gun. He still had one good engine to get home. For many genes, that’s all you need. For others, you might not even need one good copy as other genes could take over the same job.
Yes, there are some variants that you don’t want to see. Things that might indicate high likelihood of disease down the road. But even that is bound by statistical probabilities. Good news — some of those can’t be seen by this type of sequencing.
You’re absolutely right though — it can be scary. After looking at many genomes, it can be amazing that we all work as well as we do.
Additionally, we're talking about nanopore data, which is much longer reads, and so is going to be computationally a bit easier.
My current best guess is that more data than usual were of bacterial origin, but I'll have to learn a bit more bioinformatics to test that.
The fact that you got 39X raw reads is pretty good.
Also, it is usually called hg38, not hs38... I'm not sure where that came from. If you want to be super accurate, you could also use GRCh38, but there are sometimes subtle differences between the two.
https://gatk.broadinstitute.org/hc/en-us/articles/3600358909...
My mom did Nebula, which was kind of a pain, but she eventually got her results.
Their claim to sequence 100% of the genome could not be true if it was 100% DNA, as some locations like near the centromere or the telomeres are notoriousy difficult to sequence (and just impossible with the technique of alignment that they use).
It's not that bad, you can already know a lot of interesting things with a whole genome, but it won't be enough information to e.g. synthesize a copy of your DNA or be able to repair all of your adult cell's genetic damages (supposing this kind of tech exists in some future).
There are also more and more research on how the introns (DNA not in some gene) participate in the regulation of genes and are involved in many diseases.
I'd like to see some real (and affordable) 100% DNA sequencing in a near future!
Edited: s/exon/intron/
Part of the issue with centromeric or telomeric sequence is that not only is it hard to sequence (being super repetitive), little is known about what a sequence variant in such an area might mean. It's kind of a chicken-and-egg issue: it doesn't get much attention because no one knows how to interpret variants there, and since no one knows how to interpret variants there it doesn't get much attention
And given the consistency of those regions, you will have 99(.999)% of all of the information you would need to make a copy of your DNA.
There are some regions that are difficult to sequence, but the major problem isn't getting the raw sequence, but the alignment process.
If sequencing is the technique, then it's true that you can sequence anywhere in the DNA, but the technique will not always be able to give you the actual DNA sequence, in particular for centromeres or telomeres.
If sequencing meant "find out the DNA sequence", which is what people are actually buying, then it is just false that you can "sequence" anywhere in the chromosomes.
There are other techniques for getting more of the context, but that’s not the WGS we are talking about here.
It’s like copying pages from a book, but without page numbers. And some pages might be copied more than once. Or not at all (Poisson statistics FTW!). You can get fragments of stories, and can probably get the gist of the plot. But this might not give you an exact copy of the full book, in the right order.
To keep going with the analogy — what you’re asking for (telomeres and centromeres) would be the equivalent of placing every “this page left intentionally blank” page in the right place. Even though, they are all quite similar. You will be able to “copy” (sequence) each of these pages, but putting them in the right order is still difficult.
This is what I would like to see as an affordable product: get the whole sequence, so that ultimately one could theoretically re-synthesize their own chromosomes in the future. And get it now that we are still young and there is not too much damage in the DNA, information to be stored for later.
One point in what you wrote: the blank pages (introns) can be really important (like for gene expression regulation, through chromatine folding, or promoters of transcription), we just can't be sure, and that's why it is paramount (when thinking of chromosomes synthesis applications) to have it all.
About the specifics of our discussion: Again, ambiguity! Because we're saying the same thing.
When I say it does not give the sequence, I mean the whole sequence (not only local subsequences), which means including the context.
(Although the notion of context is actually meaningful only if we are talking about local subsequences, as the global sequence is the context (or includes the context, if we reduce "context" to mean the reference global sequence against which the subseqs are aligned)).
Of course with your meaning you are right.
Once we have everything, we can apply additional knowledge to this data as soon as it is discovered and without needing to do more sequencing.
Yes it is -- which is why you get that data with WGS. Seriously, the only sequence that is really difficult to get is around centromeres and telomeres (and some smaller repetitive regions). The other issues that we have with germline sequence is when there are novel structural rearrangements, but with enough depth (or a slightly different technology), we can see those too.
> Because we're saying the same thing
I don't think we are... you keep misusing some of the key terms here. Those "blank pages" weren't introns, they were the ends of the chromosomes (telomere) and the middle (centromere). Those are hard and repetitive. The rest -- the parts you'd really care about if you wanted to do some late life genetic replacement -- are largely covered.
For the context, we use a reference genome. We then look to see where the smaller subsequences align to the the reference genome. This isn't a wet-lab technique, but a dry-lab technique. With enough copies of the smaller sequences (coverage), we can establish what the underlying "full" sequence is. Given sufficient depth, this is a solved problem at this point. The only issues are depth and cost (of sequencing and processing/storage). I left this part out earlier because it wasn't what you were asking about.
We don't have a technique that you can spit in a tube and get the entire sequence of a chromosome all at once. They are simply too long for that to be practical. We do have technology to get a lot (millions) of shorter reads that we can align back to a reference (the company we are talking about in the post does this). We also have technology to get very long sequences (up to 2Mb), but they aren't as accurate as shorter reads. But, if you merge the two together, it can get you all the data you're really after here, even without a reference genome.
So, this means there is hope for getting the data required for a full DNA restoration:
- bases near the telomeres, difficult to get (i.e. difficult to locate) with standard sequencing, can be sequenced (and located) with long-sequence sequencing (but this kind of GWS cheap service doesn't use long-sequencing right?)
- for bases around centromeres, do we assume that they are surrounded by a sea of nothingness, and that relying on a unique reference genome's centromere is enough? Isn't that risky, are we really sure that individual variations do not matter for centromeres?
My aim is to imagine a future where any defects, including centromeres, could be fixed, if necessary by regenerating whole chromosomes. Using data gathered now and safely stored.
I’m skeptical that Sequencing provides the same level of a) WGS fidelity, and b) the same level of interpretation and cross reference of alleles to known or suspected pathogenic variants.