Yes, I still blog!
A quick history. As many of you will be aware, Jain et al published a fantastic paper where they produced the first de novo genome assembly of a human genome using the MinION, a portable DNA sequencer that uses nanopores to detect the sequence of single molecules. After fixing errors with Illumina, they report an accuracy of 99.8% (more on how useful that is later….)
Despite the fact I loved this paper, I was slightly frustrated that they didn’t tackle, head on, the major issues with single molecule assemblies – insertion and deletion errors. In response, I wrote my own paper, showing that indel errors are ubiquitous in both PacBio and nanopore published single molecule assemblies.
The analysis I carried out in the linked bioRxiv is fairly basic to say the least, so we have now carried out a more complex analysis using full length CDS alignments with splign. We also introduce the concept of control assemblies. GM12878 is a cell line, and as such, has accrued over time mutations and indels in areas of the genome it doesn’t use, including many genes. However, Illumina-only assemblies of NA12878 exist e.g. GCA_000185165.1 , so we can use this as a control – in other words, we can look for genes with indels in the single molecule assemblies that do not have indels in the appropriate Illumina-only assembly.
Serge Koren recently published a blog post detailing a new version of the nanopore assembly of NA12878 using only the nanopore data, in other words they didn’t use Pilon/Illumina at all, simply polished with nanopore data using the in-built canu read-correction and signal-level polishing with nanopolish. This is apparently 99.76% accurate.
So how do the two nanopore assemblies compare?
|# transcripts with indels||# genes with indels|
|Nanopore + pilon||9051||4282|
A couple of things to point out:
- We assayed around 46000 CDS transcript sequences we had evidence might be problematic
- We looked at the best, longest alignment for each CDS that was predicted to produce a protein
- We only looked at alignments including >80% of the CDS
- The above numbers are transcripts/genes that have indels in the nanopore assembly but not in the illumina assembly
We can see that both assemblies still have indel issues. The polishing with pilon has removed many, but 4282 genes remain affected. The nanopore only assembly, polished with nanopolish, has over 10,000 protein coding genes with indels.
You are shouting – show me an example!
Here is an example of a transcript aligned against an illumina assembly of NA12878: link
Here is the same transcript aligned against the nanopore-only assembly of NA12878: link
Finally, here is the same transcript in the nanpore+pilon assembly of NA12878: link
As you can see, there is no evidence from Illumina data that NA12878 has problems with this transcript. There are indels in the nanopore-only assembly, that have been fixed in the nanopore+pilon assembly.
I’m not trying to attack anyone or any technology, but we can’t fix problems if we don’t talk about them.
I remain concerned that people are publishing pacbio and nanopore assemblies without paying sufficient attention to indel errors, and our work repeatedly demonstrates that both PacBio and Nanopore assemblies suffer from the problem, even after polishing. Our own solution to this has been to fix the remaining errors manually. Do not assume that you shouldn’t be doing this!
Nanopore is a fantastic technology, but we should not overstate its accuracy nor ignore its problems.
For me, statements that entire, complex, 3Gb assemblies are “99.8%” accurate are at best completely pointless and at worst misleading. I don’t blame authors for using them, reviewers almost certainly ask for them, but they are genuinely pointless statistics.
I hope you enjoyed this blog post!