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Peter Thielen (Thielen@jhuapl.edu) from the Johns Hopkins University Applied Physics Laboratory spoke: described some metagenomic/metabarcoding tools they’ve developed. This is a follow on to our discussions last month about creation of custom databases for eDNA metabarcoding.
Title: “K-mer based read classification for eDNA data”
JHU/APL Internal research – SeqSea – an integrated approach for marine eDNA characterization.
Spoke about using oxford nanopore sequencing vs illumina sequencing for disease studies. They like oxford nanopore sequencing because it can be done at point of use rather than anchored to a lab.
Also prefer longer read lengths obtained with Oxford Nanopore
Uses Kraken for taxonomy assignments, this is way faster than BLAST. Searches for “k-mers” 31 bp long. k-mer based approaches use a pre-computed lookup table to find exact short matches of a pre-defined length, whereas BLAST and similar methods look progressively through large databases to find approximate matches of variable length. With Kraken, that database is loaded into RAM – so it’s a memory hog, but there is little to no CPU processing. There are other approaches that do more to optimize memory use (look into Centrifuge as an example). Uses lowest common ancestor automatically for k-mers that are common between species. Kraken: ultrafast metagenomic sequence classification using exact alignments | Genome Biology | Full Text (biomedcentral.com)
Challenge – Talked about some problems where phylogenetic structures can be wrong based on how they were assigned by classical means or sequence of a specific locus vs entire genomes. How well are samples placed (taxonomically) based on single-species mtDNA characterization using k-mer matching for Oxford Nanopore Sequencing (this is the technology minION instrument uses) – Classification limited to existing databases! Also thrown off by species that have close target sequences but distant phylogeny.
Peter mentioned an observation that 2 unique sequences occurred for 16S within a single bacterial culture, and this was determined not to be a sequencing error. (It came up during the discussion about Oxford nanopore sequencing being more error-prone than Illumina sequencing.) So, they had to re-isolate single colonies to start that culture over again. > from Nastassia Patin to everyone: The 16S thing could also be due to multiple gene copies in one genome
from Damian Menning to everyone: What coverage is needed for nanopore to get the correct consensus sequence? A: Oxford nanopore sequencing is more error-prone than illumina sequencing, but they aim for 20-x coverage to reliably get correct consensus sequences.
Rick L. - Anyone know of Rainbow snake eDNA markers?
from Sheena Feist to everyone: Can anyone point me in the right direction as to why LTS (light transmission spectroscopy) eDNA technologies have gone unused within recent years? Several older pubs exist for Dreissenid mussels, but appear to have gone unused since publication.
Katy Klymus suggested contacting Scott Egan, Matt Barnes and Andy Mahon are all on the LTS paper from 2015
Carol Stepien: Sheena, we did look at light transmission for zebra and quaggas and published on this by Marshall & Stepien 2019, Ecology and Evolution
Rick will be convening the steering committee this month to get started on planning the next workshop.