SEEDS Internship : DNA-based taxonomic identification of meiofauna species using in-field nanopore sequencing

In this summer project, you will work under the guidance of my lab to develop and test a rapid, fieldwork-compatible protocol for single-specimen lysis, PCR, and nanopore sequencing of individual freshwater microinvertebrates, aka meiofauna – organisms such as nematodes, tardigrades, rotifers, mites, flatworms, and more. We will test the protocol on living specimens extracted from the NHM’s wildlife garden and other London-based field sites. Our aim will be to produce barcoding data from the full ribosomal RNA cistron (18S, 28S, and both ITS markers), as well as to test “universal” COI primers. Depending on experience, sophisticated protocols such as multiplex or four-primer PCR may be attempted. Our ultimate aim is to add synthetic multiplexing indices to amplicons from all these markers, aiming to sequence 96-well plates at a time, on individual “flongle” flow cells (<1 GB output) or on a MinION flow cell, run for 2 hours at a time (using the flow cell wash kit to divide throughput over time). You will be supervised in bioinformatics aspects of the data we generate (e.g. read QC, demultiplexing, assembly, marker extraction) by my lab’s PhD student. These markers will be useful for rapid molecular identification and species delimitation, and the protocol that you validate will be used by participants in a field workshop to be undertaken this summer, which my lab is leading. This project would suit a candidate interested in invertebrate zoology, freshwater ecology, systematics, microscopy, and molecular ecology. Ideally, some prior experience with molecular protocols, especially PCR and agarose gel electrophoresis, would be beneficial. Coursework in molecular phylogenetics or invertebrate diversity/zoology will also be useful to understand the context of this work.  

Background reading (if applicable):   

Fontaneto, D., Flot, JF. & Tang, C.Q. Guidelines for DNA taxonomy, with a focus on the meiofauna. Mar Biodiv 45, 433–451 (2015). https://doi.org/10.1007/s12526-015-0319-7 

Srivathsan, A., Feng, V., Suárez, D., Emerson, B. and Meier, R. (2024), ONTbarcoder 2.0: rapid species discovery and identification with real-time barcoding facilitated by Oxford Nanopore R10.4. Cladistics, 40: 192-203. https://doi.org/10.1111/cla.12566 

Majdi, N., Schmid-Araya, J.M. & Traunspurger, W. Preface: Patterns and processes of meiofauna in freshwater ecosystems. Hydrobiologia 847, 2587–2595 (2020). https://doi.org/10.1007/s10750-020-04301-2 

Truett, Gary E., et al. "Preparation of PCR-quality mouse genomic DNA with hot sodium hydroxide and tris (HotSHOT)." Biotechniques 29.1 (2000): 52-54. 

Majdi, Nabil, et al. "Freshwater and limno-terrestrial meiofauna of the Massane Forest Reserve in the Eastern French Pyrenees." Biogeographia–The Journal of Integrative Biogeography 39.1 (2024).

A basic DBS check is required for this internship.

Work plan: Full-time

This will be a principally molecular-lab based project – your first week (e.g. June 23-27) will be an induction to the NHM’s facilities, and an introduction to meiofauna extraction and microscopy, using live meiofauna extracted from the NHM's wildlife garden.

For the next 4 weeks, we will demonstrate core molecular techniques such as:

DNA extraction using the HotSHOT protocol
PCR primer design, multiplexing strategies
PCR reaction setup
Post-PCR cleanup
Agarose gel electrophoresis
Nanopore library preparation and flow cell loading

This work will be conducted in close association with the primary supervisor (Chris Laumer), with additional support given by my lab's technician - in general each technique will be demonstrated side-by-side once, and the intern will be expected to carry out future iterations of each experiment independently (although either myself or my lab technician will always be on hand to provide support where needed). The main goal of this work is to determine which DNA extraction technique, primer-pairs/indexing design, and which amplification protocols (including multiplex PCR), are most robust to a wide variety of meiofauna species. 

With the final two weeks of the project, we will carry out some limno-terrestrial fieldwork to collect meiofauna in a real world setting (e.g. the Burnham Beeches forest), and will perform a full sequencing run on up to a 96 well plate with the specimens we discover. Finally, my current PhD student will work together with the student to demonstrate basic bioinformatics protocols for data analysis, including data demultiplexing, assembly, and BLAST-based identification and species delimitation algorithms.

The final 5 working days of the summer project will be set aside for a report/presentation writeup. 

Work plan: Part-time

In the event of an intern only being available for part time work, we would scale down the ambition, e.g. forgoing the sampling trip to Burnham Beeches and working only with fauna from the wildlife garden, and forgoing more complex experimental designs such as multiplex PCR. Depending on pace, some necessary work may also be carried out not by the student but by my lab's technician in a complementary manner.