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

Content Provider	Springer Nature : BioMed Central
Author	Karin, Benjamin R. Arellano, Selene Wang, Laura Walzer, Kayla Pomerantz, Aaron Vasquez, Juan Manuel Chatla, Kamalakar Sudmant, Peter H. Bach, Bryan H. Smith, Lydia L. McGuire, Jimmy A.
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.
Related Links	https://bmcgenomics.biomedcentral.com/counter/pdf/10.1186/s12864-023-09277-6.pdf
Ending Page	12
Page Count	12
Starting Page	1
File Format	HTM / HTML
ISSN	14712164
DOI	10.1186/s12864-023-09277-6
Journal	BMC Genomics
Issue Number	1
Volume Number	24
Language	English
Publisher	BioMed Central
Publisher Date	2023-05-02
Access Restriction	Open
Subject Keyword	Life Sciences Microarrays Proteomics Animal Genetics and Genomics Microbial Genetics and Genomics Plant Genetics and Genomics mtDNA DNA barcoding MinION LongAmp Third generation sequencing Long read sequencing Plasmid
Content Type	Text
Resource Type	Article
Subject	Biotechnology Genetics
Journal Impact Factor	3.5/2023
5-Year Journal Impact Factor	4.1/2023

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