ONT promethion for single cell human WGS?

[seq_bio]

Do let us know which way you decide in the end - Sequel or Promethion or neither. This was a very interesting discussion. In general, it looks like it's going to be tough to wean people away from Illumina as of now.
For pacbio, it looks like they need to get their costs down (throughput up) to be one platform for doing SNP, indels, CNV,SV that currently matter to a good chunk of researchers. They claim that they will increase throughput by 32x by end of 2018 fwiw - http://www.pacb.com/videos/agbt-pacb...t-lower-costs/
For ONT, accuracy appears to be a bugbear.

[WhatsOEver]

Originally Posted by gringer

Sensor noise is negligible in comparison to the shift from one base to another. All existing known base modifications produce a large current shift in the signal. Distinguishing between two different pyrimidines (i.e. C/T) is probably one of the most difficult things at the moment, because their chemical structure is so similar.

Although this becomes a little off-topic now, what do you think in this context of the genia sequencer which (more or less) specifically addresses the difficulties in distinguishing similar signals by adding tags to the bases? It seems like it might become the biggest competitor to ONT (especially with Roche behind it).

[WhatsOEver]

Originally Posted by seq_bio

Do let us know which way you decide in the end - Sequel or Promethion or neither. This was a very interesting discussion. In general, it looks like it's going to be tough to wean people away from Illumina as of now.

I will. And yes, it was for me indeed a very interesting and helpful discussion - thanks a lot to all participators!

Originally Posted by seq_bio

For pacbio, it looks like they need to get their costs down (throughput up) to be one platform for doing SNP, indels, CNV,SV that currently matter to a good chunk of researchers. They claim that they will increase throughput by 32x by end of 2018 fwiw - http://www.pacb.com/videos/agbt-pacb...t-lower-costs/
For ONT, accuracy appears to be a bugbear.

Yepp, in the end its "accuracy vs cost". Pacbio with higher accuracy but higher cost, ONT with lower accuracy but lower cost.

[nucacidhunter]

For the purpose of this thread it might worth considering 10x Genomics linked-reads as well. It has the advantage of Illumina platforms high accuracy, requires very low input DNA and currently is very cost effective in comparison to both Nanopore and PacBio, and can phase indels, SNVs and SVs over 10 Mb haplotype blocks.

[Ola]

Originally Posted by WhatsOEver

Although this becomes a little off-topic now, what do you think in this context of the genia sequencer which (more or less) specifically addresses the difficulties in distinguishing similar signals by adding tags to the bases? It seems like it might become the biggest competitor to ONT (especially with Roche behind it).

From what I have seen they can generate a ton of raw data and some sequence (bacterial genomes), but the quality per read is not impressive (yet). It relies on a polymerase which is much slower than the helicase ONT uses so yield will be lower unless they can have a much higher number of pores, and while polymerases can be highly accurate they also have a much larger variation in incorporation time so signal processing seems very challenging.

[gringer]

Originally Posted by nucacidhunter

For the purpose of this thread it might worth considering 10x Genomics linked-reads as well. It has the advantage of Illumina platforms high accuracy, requires very low input DNA and currently is very cost effective in comparison to both Nanopore and PacBio, and can phase indels, SNVs and SVs over 10 Mb haplotype blocks.

10X won't be able to resolve large tandem repeats with long repeat unit lengths, because it relies on short reads for assembly. A structure like this one, for example, won't be properly resolved:



Larger version

[gringer]

Originally Posted by WhatsOEver

Although this becomes a little off-topic now, what do you think in this context of the genia sequencer which (more or less) specifically addresses the difficulties in distinguishing similar signals by adding tags to the bases? It seems like it might become the biggest competitor to ONT (especially with Roche behind it).

That would probably work. It would make the computational side of things a bit easier, but we've by no means hit the limit of what can be done with just an electrical signal.

ONT is only the first commercial offering in what I expect will eventually be a crowded market of sequencing-by-observation devices.

[nucacidhunter]

Originally Posted by gringer

10X won't be able to resolve large tandem repeats with long repeat unit lengths, because it relies on short reads for assembly.

Amplifying single cell DNA results in fragments of 10kb average size so the input fragment length will be the limiting factor not the platform ability to sequence larger fragments. 10x linked-reads essentially amplifies large fragments to short ones for sequencing and can rebuild starting long DNA fragments.

[gringer]

If the repeat unit size is longer than the sequenced length (e.g. repeat unit size of 171bp, repeated 150 times, with a sequenced length of 125bp), and the repeat units are similar enough, then it's not possible to see from the sequence overlap alone if there is any tandemly-repeated structure at all.

A careful assembly might discover an odd increase of read coverage within a particular region, but extending that observation to a fully-resolved tandem repeat structure would be difficult.

[WhatsOEver]

Originally Posted by gringer

10X won't be able to resolve large tandem repeats with long repeat unit lengths, because it relies on short reads for assembly. A structure like this one, for example, won't be properly resolved:



Larger version

Is this human data? Could you share the raw data behind this? This might be an interesting region to evaluate our aims but I would need to check whether we are able to target this region with our approach.

[WhatsOEver]

Originally Posted by Ola

From what I have seen they can generate a ton of raw data and some sequence (bacterial genomes), but the quality per read is not impressive (yet). It relies on a polymerase which is much slower than the helicase ONT uses so yield will be lower unless they can have a much higher number of pores, and while polymerases can be highly accurate they also have a much larger variation in incorporation time so signal processing seems very challenging.

How does this fit together?
Could you tell us where you got this information from?
I would especially be interested in seeing some real data from this machine.

[gringer]

Originally Posted by WhatsOEver

Is this human data? Could you share the raw data behind this? This might be an interesting region to evaluate our aims but I would need to check whether we are able to target this region with our approach.

This is parasite DNA, but structures like this exist in human chromosomes as well, particularly in the centromeric regions. The FASTQ files associated with this (called prior to the homopolymer fixes) can be found here:

https://www.ncbi.nlm.nih.gov//sra/?term=SRP092357

I'm still working on getting the raw [signal-level] data uploaded so that people can use their own calling algorithms. SRA can't handle nanopore raw signal, so I'll need to switch to EBI/ENA for that.

I might have a look at the NA12878 genome (or Clive Brown's genome) later on to see if anything similar can be found there (adding to my list of things to do once I get back home).

[WhatsOEver]

Originally Posted by gringer

I might have a look at the NA12878 genome (or Clive Brown's genome) later on to see if anything similar can be found there (adding to my list of things to do once I get back home).

That would be great! Thanks! I'll have a look at the RepliG generated worm data in the meantime.

[ymc]

Just curious. Is it possible to use Minion to sequence a single cell without any amplification at all?

[WhatsOEver]

Originally Posted by ymc

Just curious. Is it possible to use Minion to sequence a single cell without any amplification at all?

According to the information we got from the ONT developers it is not possible. And even if it would be, you will end up with 1X (or 2X for diploid...) only with no possibility to do error correction (consensus building). You will also not be able to do any repetition - if not all DNA is sequenced (for what ever reason) it is lost. That being said, we will for sure try it sooner or later if decide to buy one

[Brian Bushnell]

Originally Posted by WhatsOEver

Why do you think so for human (or more generally multiploid organisms) WGS? For human data, we are currently unable to do whole genome reconstruction with short reads alone using the existing reference. If we create a scaffold of our genome of interest with long reads, we would still be unable to map the short reads accurately. As an example: How would a dual platform approach help me to resolve highly repetitive regions in the genome like MHC or mucins?

The combination of library types would get you highly accurate SNP and short indel calls in the nonrepetitive majority of the genome from the short reads, with minimal coverage, and SV calls across the full genome. It's also possible to combine short and long reads to accurately phase the variants called by the short reads, and potentially call variants in repetitive areas using various techniques like correcting the long reads with the short reads.

[gringer]

Originally Posted by WhatsOEver

you will end up with 1X (or 2X for diploid...) only with no possibility to do error correction (consensus building).

2X (or 4X for diploid); DNA is double-stranded, so there is a possibility of consensus sequencing using 1D².

[ymc]

Originally Posted by WhatsOEver

According to the information we got from the ONT developers it is not possible. And even if it would be, you will end up with 1X (or 2X for diploid...) only with no possibility to do error correction (consensus building). You will also not be able to do any repetition - if not all DNA is sequenced (for what ever reason) it is lost. That being said, we will for sure try it sooner or later if decide to buy one

I am interested in working on samples with around 10 human cells that share the same DNA (barring mosaicism). It will be great if you also give this scenario a try and tell us if we can get any sequences from the ten cells.

[WhatsOEver]

@Brian Agreed on everything except

potentially call variants in repetitive areas using various techniques like correcting the long reads with the short reads.

You cannot map short reads containing only repetitive sequence to a long read spanning the repetitive region, can you? If you could, you would be able to map the same short read to the reference assembly - which you can't. Or am I missing something important in your statement?

@ymc
I would start with fresh cells from the cell line for testing something that risky... but sure, I'll get back to you after we have made progression

[gringer]

mapping short reads is easier once the complete assembly exists. You can at least use probabalistic methods for mapping, and distribute reads across all the locations they could potentially map to.

Bowtie2 does this by choosing one of the candidate locations at random for each read (as long as they are equivalently incorrect, and represent the best match). It ends up being the same thing as long as there is sufficient read depth and no systematic error in the reference assembly or short reads.