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

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...

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Autores principales: 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.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10155392/
https://www.ncbi.nlm.nih.gov/pubmed/37131128
http://dx.doi.org/10.1186/s12864-023-09277-6
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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.
author_facet 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.
author_sort Karin, Benjamin R.
collection PubMed
description 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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12864-023-09277-6.
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spelling pubmed-101553922023-05-04 Highly-multiplexed and efficient long-amplicon PacBio and Nanopore sequencing of hundreds of full mitochondrial genomes 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. BMC Genomics Research Article 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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12864-023-09277-6. BioMed Central 2023-05-02 /pmc/articles/PMC10155392/ /pubmed/37131128 http://dx.doi.org/10.1186/s12864-023-09277-6 Text en © The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/ (https://creativecommons.org/publicdomain/zero/1.0/) ) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
spellingShingle Research Article
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.
Highly-multiplexed and efficient long-amplicon PacBio and Nanopore sequencing of hundreds of full mitochondrial genomes
title Highly-multiplexed and efficient long-amplicon PacBio and Nanopore sequencing of hundreds of full mitochondrial genomes
title_full Highly-multiplexed and efficient long-amplicon PacBio and Nanopore sequencing of hundreds of full mitochondrial genomes
title_fullStr Highly-multiplexed and efficient long-amplicon PacBio and Nanopore sequencing of hundreds of full mitochondrial genomes
title_full_unstemmed Highly-multiplexed and efficient long-amplicon PacBio and Nanopore sequencing of hundreds of full mitochondrial genomes
title_short Highly-multiplexed and efficient long-amplicon PacBio and Nanopore sequencing of hundreds of full mitochondrial genomes
title_sort highly-multiplexed and efficient long-amplicon pacbio and nanopore sequencing of hundreds of full mitochondrial genomes
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10155392/
https://www.ncbi.nlm.nih.gov/pubmed/37131128
http://dx.doi.org/10.1186/s12864-023-09277-6
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