Feb 6, 2017

Oxford MinION 1D and 2D reads

We have been testing out the Oxford MinION DNA sequencing machine to see what it can contribute to larger de novo sequencing projects.  Most of the posted data so far come from small genomes where moderate coverage is more easily obtained.  Recent publications claim improved sequence quality for Oxfort MinION.

We are working on a new de novo sequence for the little skate shark ((Leucoraja erinacea), an interesting system to study developmental biology.  The skate has a BIG genome, estimated at 4 Gb (bigger than human), so this is going to be a difficult project. The existing skate genome in Genbank and the SkateBase website is not in very good shape (3 million contigs with  N50 size of 665 bp).

We got a couple of preliminary Oxford MinION reads from skate DNA - not nearly enough coverage to make a dent in this project, just having a look at the data. Oxford produces two kinds of data (in their own annoying FAST5 format, but I won't rant about that right now), single pass 1D and double pass 2D.  [My Bioinformatics programmer Yuhan Hao did this analysis.] Here is what our data looks like.


So the 1D reads are really long - some more than 50 kb. The 2D reads are mostly 2-10 kb.  The SkateBase has a complete contig of the mitochondrial genome, so we were able to align the Oxford sequences to this as a reference. Coverage was low, but we do have some regions where both 1D and 2D reads match the reference. What we can see is that the 1D reads have a lot of SNPs vs the reference, while the 2D reads have very few SNPs- so it is clear that the 2D reads have been successfully error corrected.  Strangely, both the 1D and 2D reads have a lot of insertion-deletion errors (several per hundred bases) compared to the reference, and in fact they do not match each other - so we consider these to all be novel, uncorrected errors.







We also ran a standard Illumina whole genome shotgun sequencing run for the skate genome, which we aligned to the mitochondrial reference. With this data, we can see a small number of Oxford 2D SNPs shared by hundreds of Illumina reads, others not. None of the indels are supported by our Illumina reads.


Other investigators have had poor quality Oxoford sequences. With more coverage, we may be able to use the Oxoford reads as scaffolds for our de novo assembly. It may be possible to use Illumina reads for error correction, and mark all uncorrected areas of the Oxford sequences as low quality, but that is not the usual method for submitting draft genomes to Genbank.

Jan 17, 2017

GenomeWeb reports on updated Coffee Beetle genome

A nice review of my 2015 Coffee Beetle paper in GenomeWeb today.
My genome had 163 million bases and 19,222 predicted protein-coding genes. I am very pleased to learn that a revised version of the draft genome sequence (from a group in Columbia) contains 160 million bases and 22,000 gene models. They also confirm the 12 horizontally transferred genes that I identified.

Coffee Pest, Plant Genomes Presented at PAG Conference

Researchers from the USDA's Agricultural Research Service, New York University, King Abdullah University of Science and Technology, and elsewhere published information on a 163 million base draft genome for the coffee berry borer in the journal Scientific Reports in 2015.
That genome assembly, produced with Illumina HiSeq 2000 reads, housed hundreds of small RNAs and an estimated 19,222 protein-coding genes, including enzymes, receptors, and transporters expected to contribute to coffee plant predation, pesticide response, and defense against potential pathogens. It also provided evidence of horizontal gene transfer involving not only mannanase, but several other bacterial genes as well.
At the annual Plant and Animal Genomes meeting here this week, National Center for Coffee Research (Cenicafe) scientist Lucio Navarro provided an update on efforts to sequence and interpret the coffee berry borer genome during a session on coffee genomics. For their own recent analyses, Navarro and his colleagues upgraded an earlier version of a coffee berry borer genome that had been generated by Roche 454 FLX sequencing, using Illumina short reads from male and female coffee berry borers to produce a consensus assembly spanning around 160 million bases. The assembly is believed to represent roughly 96 percent of the insect's genome.
In addition to producing a genome with improved contiguity levels, he reported, members of that team also combined 454 and Illumina reads to get consensus transcriptomes for the beetle. With these and other data, they identified almost 22,000 gene models, novel transposable element families, and their own evidence of horizontal gene transfer.