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Abstract Detail


Rothfels, Carl [1], Pryer, Kathleen [2], Li, Fay-Wei [3].

Next-generation polyploid phylogenetics: Low-cost, high-throughput resolution of hybrid polyploid complexes using PacBio and PURC.

Nuclear sequence data are critically important for phylogenetic inference in plants, where polyploidy, hybridization, and gene-family evolution can be especially confounding. These data remain difficult and expensive to generate, however, especially for those taxonomic groups with few available genomic resources, or where multiple, distinct gene copies (homeologs or paralogs) are often present in individual accessions. Here we describe an amplicon-sequencing method for generating long (~1kb) sequences of multiple nuclear loci from multiple mostly-polyploid accessions. Our protocol utilizes the PacBio sequencing platform to generate data quickly and cheaply. It has limited up-front investment of time or money, does not require any downstream sequence phasing or assembly, and yields all the copies (alleles, homeologs, or paralogs) amplified by a given primer pair, for each accession. Our newly-developed bioinformatics package PURC (“Pipeline for Untangling Reticulate Complexes”) then takes the raw sequence reads as input and infers the true biological sequences, producing alignments for each locus, with each sequence labeled according to its source accession and its depth of sequencing coverage. PURC trims primer and barcode sequences from the data and, via an iterative clustering process, corrects PCR and sequencing errors and also removes PCR-mediated recombinant sequences (chimeras). PURC then matches sequences to source accessions based on sequence similarity as well as barcode sequence, allowing individual barcodes to be used multiple times within a single run. Using data from four nuclear loci for a sample of mostly polyploid accessions from the fern family Cystopteridaceae, we demonstrate the efficacy of this combined wet lab/bioinformatics approach, and also its prospects for yielding novel evolutionary insights.

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1 - University of California Berkeley, University Herbarium and Dept. of Integrative Biology, Berkeley, CA, 94720, USA
2 - Duke University, Biology, Durham, NC, 27708, USA
3 - University of California Berkeley, Integrative Biology, Berkeley, CA, 94720, USA

High-Throughput Sequencing
Next generation sequencing
reticulate evolution
low-copy nuclear markers
species tree
chimeric sequences
phylogenetic networks.

Presentation Type: Oral Paper
Session: 16, Phylogenomics II
Location: 202/Savannah International Trade and Convention Center
Date: Tuesday, August 2nd, 2016
Time: 8:15 AM
Number: 16002
Abstract ID:543
Candidate for Awards:Margaret Menzel Award

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