Highly-multiplexed and efficient long-amplicon PacBio and Nanopore sequencing of hundreds of full mitochondrial genomes

Benjamin R Karin; Selene Arellano; Laura Wang; Kayla Walzer; Aaron Pomerantz; Juan Manuel Vasquez; Kamalakar Chatla; Peter H Sudmant; Bryan H Bach; Lydia L Smith; Jimmy A McGuire

doi:10.1186/s12864-023-09277-6

Highly-multiplexed and efficient long-amplicon PacBio and Nanopore sequencing of hundreds of full mitochondrial genomes

BMC Genomics. 2023 May 2;24(1):229. doi: 10.1186/s12864-023-09277-6.

Authors

Benjamin R Karin^{1

2}, Selene Arellano³, Laura Wang³, Kayla Walzer³, Aaron Pomerantz³, Juan Manuel Vasquez³, Kamalakar Chatla³, Peter H Sudmant^{3

4}, Bryan H Bach^{5

6

7}, Lydia L Smith⁵, Jimmy A McGuire^{3

5}

Affiliations

¹ Department of Integrative Biology, Valley Life Sciences Building, University of California, Berkeley, CA, 94708, USA. benkarin@berkeley.edu.
² Museum of Vertebrate Zoology, University of California, Berkeley, CA, USA. benkarin@berkeley.edu.
³ Department of Integrative Biology, Valley Life Sciences Building, University of California, Berkeley, CA, 94708, USA.
⁴ Center for Computational Biology, University of California, Berkeley, CA, USA.
⁵ Museum of Vertebrate Zoology, University of California, Berkeley, CA, USA.
⁶ Department of Environmental Science, Policy, and Management, University of California, Berkeley, CA, USA.
⁷ Innovative Genomics Institute, University of California, Berkeley, CA, USA.

Abstract

Background: Mitochondrial genome sequences have become critical to the study of biodiversity. Genome skimming and other short-read based methods are the most common approaches, but they are not well-suited to scale up to multiplexing hundreds of samples. Here, we report on a new approach to sequence hundreds to thousands of complete mitochondrial genomes in parallel using long-amplicon sequencing. We amplified the mitochondrial genome of 677 specimens in two partially overlapping amplicons and implemented an asymmetric PCR-based indexing approach to multiplex 1,159 long amplicons together on a single PacBio SMRT Sequel II cell. We also tested this method on Oxford Nanopore Technologies (ONT) MinION R9.4 to assess if this method could be applied to other long-read technologies. We implemented several optimizations that make this method significantly more efficient than alternative mitochondrial genome sequencing methods.

Results: With the PacBio sequencing data we recovered at least one of the two fragments for 96% of samples (~ 80-90%) with mean coverage ~ 1,500x. The ONT data recovered less than 50% of input fragments likely due to low throughput and the design of the Barcoded Universal Primers which were optimized for PacBio sequencing. We compared a single mitochondrial gene alignment to half and full mitochondrial genomes and found, as expected, increased tree support with longer alignments, though whole mitochondrial genomes were not significantly better than half mitochondrial genomes.

Conclusions: This method can effectively capture thousands of long amplicons in a single run and be used to build more robust phylogenies quickly and effectively. We provide several recommendations for future users depending on the evolutionary scale of their system. A natural extension of this method is to collect multi-locus datasets consisting of mitochondrial genomes and several long nuclear loci at once.

Keywords: DNA barcoding; Long read sequencing; LongAmp; MinION; Plasmid; Third generation sequencing; mtDNA.

MeSH terms

Biodiversity
Genome, Mitochondrial*
High-Throughput Nucleotide Sequencing / methods
Nanopore Sequencing*
Nanopores*
Sequence Analysis, DNA / methods

Grants and funding

Pacific Biosciences SMRT Grant/Pacific Biosciences SMRT Grant