Abstract Detail

Mycology

Berta-Thompson, Jessie W. [1], Olds, Gary [1], Loucks, Justin [1], Levy, Rick [1], Wilson, Andrew [1].

What’s in a fungarium specimen? Exploring variation in deep short-read sequencing of preserved fungal specimen nrITS2 barcodes.

DNA sequencing from fungarium specimens represents a powerful resource for fungal systematics and biodiversity work, linking traditional macrofungal taxonomy to the molecular. Previously limited to sequencing a few marker genes, museum specimen sequencing now benefits from genomic and high-throughput approaches. To expand fungal nrITS barcode sequencing to more untapped preserved specimens, our group has developed a high-throughput Illumina protocol that enables affordable sequencing of the variable nrITS2 region (250-400 bp) with universal fungal primers for hundreds of specimens at once. This approach generates hundreds to thousands of raw sequences per specimen, depending on multiplexing choices and sequencing depth. Each specimen's sequence pool contains distinct sequences for nrITS2 variants within a genome, both SNPs and indels, as well as erroneous sequences and sequences from associated or contaminating fungi. While the primary purpose of these efforts is to obtain a specimen's singular taxonomic barcode, the multiple sequences provided by this method present new challenges and opportunities. Here we present an exploration of what can be mined from a single specimen's pool of sequences, using initial datasets from Southern Rocky Mountain fungal specimens chosen to address targeted taxonomic questions and to support a proof-of-concept for our methods method across many taxa. Our sequence processing approach uses basic short-read tools to quantify, assess, and prepare the raw data and the dada2 pipeline to remove chimeras and reduce sequencing errors.   These steps simplify the data to tens of unique sequences for each specimen, still a complex pool. Read abundance counts associated with each sequence at this stage can be used to extract the dominant target sequence(s). We can also explore the off-target sequences from other fungi, at lower abundance in the read counts. Some of these tag-along organisms are present across many specimens while others are rare. These have the potential to provide insights about specimen storage and associations between micro and macrofungi. Finally, we present preliminary detections of intragenomic variants with this method, and the types, patterns and frequency of these variants in our dataset. In future, we hope this technique will prove useful for unlocking more of the potential hidden in collections while adding nuance to our understanding of fungal specimen nrITS sequences through deep sequencing.

1 - Denver Botanic Gardens, 1007 York St., Denver, CO, 80206, United States

Keywords:
Mycology
herbarium specimen
Fungarium
high throughput sequencing
intragenomic variation.

Presentation Type: Poster
Session: MYP1, Mycology Posters I
Location: /
Date: Monday, July 19th, 2021
Time: 5:00 PM(EDT)
Number: MYP1019
Abstract ID:857
Candidate for Awards:None