Recently I published a blog post about a fairly simple test to find out whether you have “short” protein predictions in your bacterial genomes, and predicted that some of these short peptides may be the result of unresolved errors in long-read, single molecule assemblies.

Perhaps not surprisingly, there was a reaction from the PacBio community over this, and here is my response.

Before I begin, I just want to say that, whilst most people see me as some kind of Nanopore fan-boy, the reality is I am a fan of cool technology and that includes PacBio.  The hard facts are that I have spent around £200k on PacBio sequencing over the last 18 months, and about £20k on nanopore in the same time period.  I also encouraged our core to buy a PacBio Sequel.  So I am not anti-Pacbio.  I am, however, anti-bullshit 😉

In addition, my blog post wasn’t about problems with technology per se, it was about problems with people.  If you don’t know errors are there, you might not correct them, and if you believe company hype (for example that PacBio data is Q50 after polishing) you might believe your assembly is perfect.

It isn’t.  They never are.

Let’s dive into some data.  The three PacBio genomes I chose in the last blog post are:

AP018165	Mycobacterium stephanolepidis 
CP025317	Escherichia albertii 
ERS530415	Yersinia enterocolitica

if you search GenBank genomes for these, there is only one Mycobacterium stephanolepidis genome, but there are 39 Escherichia albertii genomes and there are 176 Yersinia enterocolitica.  Many of these will be sequenced on different technologies, which allows us to do a comparison within a species.

Escherichia albertii 

I downloaded the 39 genomes from GenBank, and ran the process I described last week.  For the complete genomes, this is what the data look like, ordered from lowest number of short peptides, to greatest:

Accession Technology Contigs # proteins # short
GCA_001549955.1 Sanger+454 1 4299 19
GCA_002285455.1 Sanger+454 1 4157 19
GCA_002285475.1 Sanger+454 1 4295 28
GCA_002741375.1 PacBio 1 (plus 6 plasmids) 5126 79
GCA_002895205.1 Illumina+454+Ion 1 4423 83
GCA_000512125.1 PacBio 1 4482 106
GCA_002872455.1 PacBio 1 (plus 3 plasmids) 4984 124

Sure is unlucky that PacBio assemblies are at the bottom of the table isn’t it?  Of course, Sanger is the gold standard, and many will be asking what the Illumina assemblies look like.

Let’s look at the next ten genomes, which are not complete, but are the least fragmented:

Accession Technology Contigs # proteins # short
GCA_002109845.1 454 25 4758 93
GCA_001514945.1 Illumina 43 4237 82
GCA_002563295.1 Ion 44 4531 143
GCA_001514965.1 Illumina 50 4390 86
GCA_001515045.1 Illumina 53 4480 110
GCA_001515005.1 Illumina 59 4963 117
GCA_001514645.1 Illumina 63 4470 84
GCA_001514685.1 Illumina 70 4209 68
GCA_001514925.1 Illumina 73 4464 84
GCA_001514905.1 Illumina 78 4622 113

What I think is worth pointing out here is that the PacBio genomes in the first table, which are complete, have about the same number of short proteins as the Illumina and 454 assemblies in the second table, which are fragmented.  We would usually expect fragmented assemblies to have more short proteins because the contig ends would interupt ORFs.   Indeed, compared to Sanger complete assemblies, the fragmented assemblies do have more short proteins.

They just don’t have more than the PacBio complete assemblies.  Odd that.  If there were no uncorrected errors in the PacBio assemblies, they would be more like the Sanger assemblies than the Illumina ones.

Yersinia enterocolitica

Of the 176 GenBank genomes for this species, my automated script could only detect sequencing technology for 35 of them.  Here are the 21 that have a claim to be complete or near-complete (<5 contigs)

Accession Technology contigs # proteins # short
GCA_000834195.1 Illumina+454 2 4161 17
GCA_000987925.1 PacBio 2 4340 19
GCA_001304755.1 PacBio 2 4344 20
GCA_002082275.2 PacBio+Illumina 1 4067 31
GCA_002554625.2 PacBio+Illumina 1 4384 35
GCA_001305635.1 Illumina 2 4162 35
GCA_000834795.1 PacBio+Illumina 2 4259 36
GCA_000755045.1 Illumina+454 2 4226 44
GCA_000834735.1 PacBio+Illumina 2 4138 48
GCA_000755055.1 Illumina+454 1 4095 53
GCA_000754975.1 Illumina+454 2 4083 53
GCA_000597945.2 Illumina+454 3 4640 59
GCA_000754985.1 Illumina+454 3 4282 65
GCA_002083285.2 PacBio+Illumina 4 4198 76
GCA_002082245.2 PacBio+Illumina 2 4521 106
GCA_001708575.1 PacBio 3 4673 221
GCA_001708615.1 PacBio 3 4615 224
GCA_001708635.1 PacBio 3 4659 224
GCA_001708655.1 PacBio 3 4654 226
GCA_001708555.1 PacBio 3 4674 230
GCA_001708595.1 PacBio 2 4733 235

Slightly different story here, in that the PacBio (and PacBio hybrid) genomes appear to have some of the lowest number of predicted short proteins.  This is what people now expect when they see PacBio bacterial genomes.  However, there are also six PacBio genomes at the bottom of the table, and so I don’t think you can really look at this data and think there isn’t a problem.  It’s possible that those six just happen to be the strains of Yersinia enterocolitica that have undergone the most pseudogenisation, but I don’t think so.



Let’s get some things straight

  • I know this is an incomplete analysis and obviously more work needs to be done
  • If I personally wanted a perfect microbial genome, I would probably use PacBio+Illumina
  • I have nothing against PacBio
  • Nanopore aren’t in this list because they’re not in the species I chose, but I am sure they would also have significant problems

 

As I said above, it’s not that PacBio has a problem per se, it’s that people have a problem.  Yes, many errors are correctable with Quiver, Arrow and Pilon; often multiple rounds are necessary and you still won’t catch everything.

But not everyone knows that, and it’s clear to me from the above that many people are still pumping out poor quality, uncorrected, indel-ridden PacBio genomes.

The same is true for Nanopore, I have no doubt.

Let’s stop bullshitting.  These technologies have problems.  It doesn’t mean they are bad technologies, anything but – both PacBio and Nanopore have been transformational.  There is no need for bullshit.

“With great power comes great responsibility” – in this case, the responsibility is two-fold.  One, stop contaminating public databases with sh*t assemblies; and two, stop bullshitting that this isn’t a problem for your favourite technology.  That helps no-one.