A highly flexible and repeatable genotyping method for aquaculture studies based on target amplicon sequencing using next-generation sequencing technology

Sato, Mana; Hosoya, S.; Yoshikawa, Sota; Ohki, Shun; Kobayashi, Yuki; Itou, Takuya; Kikuchi, Kiyoshi

doi:10.1038/s41598-019-43336-x

Cited by 36 publications

(32 citation statements)

References 54 publications

(68 reference statements)

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“…1E) (Campbell et al, 2015; Lepais et al, 2019; Natesh et al, 2019) while others performed the cleanup after both PCR steps (Fig. 1B and E) (Aykanat, Lindqvist, Pritchard, & Primmer, 2016; Onda, Takahagi, Shimizu, Inoue, & Mochida, 2018; Sato et al, 2019). Performing only one cleanup step saves on cost and labor but may have downstream effects on the final results such as higher amounts of non-specific PCR products produced (Aykanat et al, 2016).…”

Section: Introductionmentioning

confidence: 99%

More affordable and effective noninvasive SNP genotyping using high-throughput amplicon sequencing

Eriksson

Ruprecht

Levi

2019

Preprint

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5Non-invasive genotyping methods have become key elements of wildlife research over the last 6 two decades, but their widespread adoption is limited by high costs, low success rates, and high 7 error rates. The information lost when genotyping success is low may lead to decreased precision 8 in animal population densities which could misguide conservation and management actions. Single 9 nucleotide polymorphisms (SNPs) provide a promising alternative to traditionally used 10 microsatellites as SNPs allow amplification of shorter DNA fragments, are less prone to 11 genotyping errors, and produce results that are easily shared among laboratories. Here, we outline 12 a detailed protocol for cost-effective and accurate noninvasive SNP genotyping using highly 13 multiplexed amplicon sequencing optimized for degraded DNA. We validated this method for 14 individual identification by genotyping 216 scats, 18 hairs and 15 tissues from coyotes (Canis 15 latrans). Our genotyping success rate for scat samples was 93%, and 100% for hair and tissue, 16 representing a substantial increase compared to previous microsatellite-based studies at a cost of 17 under $5 per PCR replicate (excluding labor). The accuracy of the genotypes was further 18 corroborated in that genotypes from scats matching known, GPS-collared coyotes were always 19 located within the territory of the known individual. We also show that different levels of 20 multiplexing produced similar results, but that PCR product cleanup strategies can have 21 2 substantial effects on genotyping success. By making noninvasive genotyping more affordable, 22 accurate, and efficient, this research may allow for a substantial increase in the use of 23 noninvasive methods to monitor and conserve free-ranging wildlife populations. 24 Keywords 25 Fecal DNA, individual identification, next-generation sequencing, noninvasive samples, single 26 nucleotide polymorphisms, SNP genotyping. 27 28 29 48 rates when using fragmented DNA from noninvasive sources (Morin, Luikart, & Wayne, 2004).49 Unlike microsatellite genotyping, which use size-based allele determination, SNP alleles are 50 provided directly in the DNA sequence, which facilitates automated allele calling and results that 51 are easily shared among laboratories and research groups.52 4 Recent SNP genotyping approaches for noninvasive samples include the Fluidigm 53 Dynamic Array platform (Fluidigm Corp) and the MassARRAY platform from Sequenom. The 54 Fluidigm platform in particular has produced high genotyping success and low error rates for 55 both fecal and hair samples (Kraus et al., 2015; Von Thaden et al., 2017). However, these 56 techniques require specialized equipment often rendering them prohibitively expensive for 57 wildlife research (Andrews, De Barba, Russello, & Waits, 2018; Carroll et al., 2018). A largely 58 unexplored strategy is to amplify many SNPs together in multiplex PCR and sequence them 59 using high-throughput sequencers. Such genotyping by amplicon sequencing (GBAS) is both 60 cost-effective, a...

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Section: Introductionmentioning

confidence: 99%

More affordable and effective noninvasive SNP genotyping using high-throughput amplicon sequencing

Eriksson

Ruprecht

Levi

2019

Preprint

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“…In this study, we could not detect significant SNPs from GWAS. Even with the small SNP panel and small sample size, strong effect SNPs (the sex-determining SNP) could be detected in a cultured population of the tiger pufferfish 39 . Therefore, our GWAS result suggests the parasitic resistance is controlled by a large number of quantitative trait locus (QTL) with small or moderate effects, and marker-assisted selection is not feasible.…”

Section: Discussionmentioning

confidence: 99%

“…We genotyped genome-wide SNPs of each individual using AmpliSeq 39 . The MiSeq sequencing generated an average of 174,870 (± 83,576 S.D.)…”

Section: Genotypingmentioning

confidence: 99%

Genomic selection for heterobothriosis resistance concurrent with body size in the tiger pufferfish, Takifugu rubripes

Lin

Sato

Mizuno

et al. 2020

Sci Rep

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Parasite resistance traits in aquaculture species often have moderate heritability, indicating the potential for genetic improvements by selective breeding. However, parasite resistance is often synonymous with an undesirable negative correlation with body size. In this study, we first tested the feasibility of genomic selection (GS) on resistance to heterobothriosis, caused by the monogenean parasite Heterobothrium okamotoi, which leads to huge economic losses in aquaculture of the tiger pufferfish Takifugu rubripes. Then, using a simulation study, we tested the possibility of simultaneous improvement of parasite resistance, assessed by parasite counts on host fish (HC), and standard length (SL). Each trait showed moderate heritability (square-root transformed HC: h2 = 0.308 ± 0.123, S.E.; SL: h2 = 0.405 ± 0.131). The predictive abilities of genomic prediction among 12 models, including genomic Best Linear Unbiased Predictor (GBLUP), Bayesian regressions, and machine learning procedures, were also moderate for both transformed HC (0.248‒0.344) and SL (0.340‒0.481). These results confirmed the feasibility of GS for this trait. Although an undesirable genetic correlation was suggested between transformed HC and SL (rg = 0.228), the simulation study suggested the desired gains index can help achieve simultaneous genetic improvements in both traits.

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“…Such approach is commonly called "multiplex PCR" and, for example, was applied as a genotyping tool for 192 loci on 2068 DNA samples of the fish Oncorhynchus mykiss in a single Illumina HiSeq 1500 run [137]. A multiplex PCR of >3000 primer pairs yielded a set of 2655 reproducible SNPs on two pooled samples of fugu fish (Takifugu rubripes), each with the same 326 individually tagged fishes [139].…”

Section: Amplicon Sequencingmentioning

confidence: 99%

DNA-Based Assessment of Genetic Diversity in Grassland Plant Species: Challenges, Approaches, and Applications

2019

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Grasslands are wide-spread, multi-species ecosystems that provide many valuable services. Plant genetic diversity (i.e., the diversity within species) is closely linked to ecosystem functioning in grasslands and constitutes an important reservoir of genetic resources that can be used to breed improved cultivars of forage grass and legume species. Assessing genetic diversity in grassland plant species is demanding due to the large number of different species and the level of resolution needed. However, recent methodological advances could help in tackling this challenge at a larger scale. In this review, we outline the methods that can be used to measure genetic diversity in plants, highlighting their strengths and limitations for genetic diversity assessments of grassland plant species, with a special focus on forage plants. Such methods can be categorized into DNA fragment, hybridization array, and high-throughput sequencing (HTS) methods, and they differ in terms of resolution, throughput, and multiplexing potential. Special attention is given to HTS approaches (i.e., plastid genome skimming, whole genome re-sequencing, reduced representation libraries, sequence capture, and amplicon sequencing), because they enable unprecedented large-scale assessments of genetic diversity in non-model organisms with complex genomes, such as forage grasses and legumes. As no single method may be suited for all kinds of purposes, we also provide practical perspectives for genetic diversity analyses in forage breeding and genetic resource conservation efforts.

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A highly flexible and repeatable genotyping method for aquaculture studies based on target amplicon sequencing using next-generation sequencing technology

Cited by 36 publications

References 54 publications

More affordable and effective noninvasive SNP genotyping using high-throughput amplicon sequencing

More affordable and effective noninvasive SNP genotyping using high-throughput amplicon sequencing

Genomic selection for heterobothriosis resistance concurrent with body size in the tiger pufferfish, Takifugu rubripes

DNA-Based Assessment of Genetic Diversity in Grassland Plant Species: Challenges, Approaches, and Applications

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