Challenges and Approaches to Genotyping Repetitive DNA
Individuals within a species can exhibit vast variation in copy number of repetitive DNA elements. This variation may contribute to complex traits such as lifespan and disease, yet it is only infrequently considered in genotype-phenotype associations. Although the possible importance of copy number...
Main Authors: | , , , , , , , , , |
---|---|
Format: | Article |
Language: | English |
Published: |
Oxford University Press
2020-01-01
|
Series: | G3: Genes, Genomes, Genetics |
Subjects: | |
Online Access: | http://g3journal.org/lookup/doi/10.1534/g3.119.400771 |
id |
doaj-2aa0e1c9e4854b5a8b31e14929ff203f |
---|---|
record_format |
Article |
spelling |
doaj-2aa0e1c9e4854b5a8b31e14929ff203f2021-07-02T13:12:10ZengOxford University PressG3: Genes, Genomes, Genetics2160-18362020-01-0110141743010.1534/g3.119.40077136Challenges and Approaches to Genotyping Repetitive DNAElizabeth A. MortonAshley N. HallElizabeth KwanCalvin MokKonstantin QueitschVivek NandakumarJohn StamatoyannopoulosBonita J. BrewerRobert WaterstonChristine QueitschIndividuals within a species can exhibit vast variation in copy number of repetitive DNA elements. This variation may contribute to complex traits such as lifespan and disease, yet it is only infrequently considered in genotype-phenotype associations. Although the possible importance of copy number variation is widely recognized, accurate copy number quantification remains challenging. Here, we assess the technical reproducibility of several major methods for copy number estimation as they apply to the large repetitive ribosomal DNA array (rDNA). rDNA encodes the ribosomal RNAs and exists as a tandem gene array in all eukaryotes. Repeat units of rDNA are kilobases in size, often with several hundred units comprising the array, making rDNA particularly intractable to common quantification techniques. We evaluate pulsed-field gel electrophoresis, droplet digital PCR, and Nextera-based whole genome sequencing as approaches to copy number estimation, comparing techniques across model organisms and spanning wide ranges of copy numbers. Nextera-based whole genome sequencing, though commonly used in recent literature, produced high error. We explore possible causes for this error and provide recommendations for best practices in rDNA copy number estimation. We present a resource of high-confidence rDNA copy number estimates for a set of S. cerevisiae and C. elegans strains for future use. We furthermore explore the possibility for FISH-based copy number estimation, an alternative that could potentially characterize copy number on a cellular level.http://g3journal.org/lookup/doi/10.1534/g3.119.400771ribosomal dnarepetitive dnawhole genome sequencingcopy number variation |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Elizabeth A. Morton Ashley N. Hall Elizabeth Kwan Calvin Mok Konstantin Queitsch Vivek Nandakumar John Stamatoyannopoulos Bonita J. Brewer Robert Waterston Christine Queitsch |
spellingShingle |
Elizabeth A. Morton Ashley N. Hall Elizabeth Kwan Calvin Mok Konstantin Queitsch Vivek Nandakumar John Stamatoyannopoulos Bonita J. Brewer Robert Waterston Christine Queitsch Challenges and Approaches to Genotyping Repetitive DNA G3: Genes, Genomes, Genetics ribosomal dna repetitive dna whole genome sequencing copy number variation |
author_facet |
Elizabeth A. Morton Ashley N. Hall Elizabeth Kwan Calvin Mok Konstantin Queitsch Vivek Nandakumar John Stamatoyannopoulos Bonita J. Brewer Robert Waterston Christine Queitsch |
author_sort |
Elizabeth A. Morton |
title |
Challenges and Approaches to Genotyping Repetitive DNA |
title_short |
Challenges and Approaches to Genotyping Repetitive DNA |
title_full |
Challenges and Approaches to Genotyping Repetitive DNA |
title_fullStr |
Challenges and Approaches to Genotyping Repetitive DNA |
title_full_unstemmed |
Challenges and Approaches to Genotyping Repetitive DNA |
title_sort |
challenges and approaches to genotyping repetitive dna |
publisher |
Oxford University Press |
series |
G3: Genes, Genomes, Genetics |
issn |
2160-1836 |
publishDate |
2020-01-01 |
description |
Individuals within a species can exhibit vast variation in copy number of repetitive DNA elements. This variation may contribute to complex traits such as lifespan and disease, yet it is only infrequently considered in genotype-phenotype associations. Although the possible importance of copy number variation is widely recognized, accurate copy number quantification remains challenging. Here, we assess the technical reproducibility of several major methods for copy number estimation as they apply to the large repetitive ribosomal DNA array (rDNA). rDNA encodes the ribosomal RNAs and exists as a tandem gene array in all eukaryotes. Repeat units of rDNA are kilobases in size, often with several hundred units comprising the array, making rDNA particularly intractable to common quantification techniques. We evaluate pulsed-field gel electrophoresis, droplet digital PCR, and Nextera-based whole genome sequencing as approaches to copy number estimation, comparing techniques across model organisms and spanning wide ranges of copy numbers. Nextera-based whole genome sequencing, though commonly used in recent literature, produced high error. We explore possible causes for this error and provide recommendations for best practices in rDNA copy number estimation. We present a resource of high-confidence rDNA copy number estimates for a set of S. cerevisiae and C. elegans strains for future use. We furthermore explore the possibility for FISH-based copy number estimation, an alternative that could potentially characterize copy number on a cellular level. |
topic |
ribosomal dna repetitive dna whole genome sequencing copy number variation |
url |
http://g3journal.org/lookup/doi/10.1534/g3.119.400771 |
work_keys_str_mv |
AT elizabethamorton challengesandapproachestogenotypingrepetitivedna AT ashleynhall challengesandapproachestogenotypingrepetitivedna AT elizabethkwan challengesandapproachestogenotypingrepetitivedna AT calvinmok challengesandapproachestogenotypingrepetitivedna AT konstantinqueitsch challengesandapproachestogenotypingrepetitivedna AT viveknandakumar challengesandapproachestogenotypingrepetitivedna AT johnstamatoyannopoulos challengesandapproachestogenotypingrepetitivedna AT bonitajbrewer challengesandapproachestogenotypingrepetitivedna AT robertwaterston challengesandapproachestogenotypingrepetitivedna AT christinequeitsch challengesandapproachestogenotypingrepetitivedna |
_version_ |
1721329076430438400 |