Targeted multiplex next‐generation sequencing: advances in techniques of mitochondrial and nuclear DNA sequencing for population genomics

Abstract

Next-generation sequencing (NGS) is emerging as an efficient and cost-effective tool in population genomic analyses of nonmodel organisms, allowing simultaneous resequencing of many regions of multi-genomic DNA from multiplexed samples. Here, we detail our synthesis of protocols for targeted resequencing of mitochondrial and nuclear loci by generating indexed genomic libraries for multiplexing up to 100 individuals in a single sequencing pool, and then enriching the pooled library using custom DNA capture arrays. Our use of DNA sequence from one species to capture and enrich the sequencing libraries of another species (i.e. cross-species DNA capture) indicates that efficient enrichment occurs when sequences are up to about 12% divergent, allowing us to take advantage of genomic information in one species to sequence orthologous regions in related species. In addition to a complete mitochondrial genome on each array, we have included between 43 and 118 nuclear loci for low-coverage sequencing of between 18 kb and 87 kb of DNA sequence per individual for single nucleotide polymorphisms discovery from 50 to 100 individuals in a single sequencing lane. Using this method, we have generated a total of over 500 whole mitochondrial genomes from seven cetacean species and green sea turtles. The greater variation detected in mitogenomes relative to short mtDNA sequences is helping to resolve genetic structure ranging from geographic to species-level differences. These NGS and analysis techniques have allowed for simultaneous population genomic studies of mtDNA and nDNA with greater genomic coverage and phylogeographic resolution than has previously been possible in marine mammals and turtles.

DNA Sequencing as a Tool to Monitor Marine Ecological Status

“…Such work is buttressed by genomic-based studies which continue to advance, in part, by increased access to and application of DNA sequencing technologies (Cammen et al, 2016;Leslie and Morin, 2016). Declining costs encourage sequencing as an alternative to traditional biodiversity monitoring (Bourlat et al, 2013) and supports continued advances in conservation biology (Wallace et al, 2010;Hancock-Hanser et al, 2013). Although information derived from DNA sequencing is yet to be formally included in current marine status assessment programs (Bourlat et al, 2013), there is widespread recognition of the importance of this approach, and an increasing number of projects (Table 2) generate sequence-based biodiversity assessments.…”

Advances in the Application of Genetics in Marine Turtle Biology and Conservation

“…Informative nuclear markers have also now been developed for all marine turtle species, offering finer-scale resolution in some biological contexts, and applicability to research questions about the male component of populations. This has included informative microsatellites (see Glossary; e.g., Aggarwal et al, 2004;Jensen et al, 2006;Shamblin et al, 2007Shamblin et al, , 2009Shamblin et al, , 2012cAlstad et al, 2011;Roden and Dutton, 2011) and more recently, single nucleotide polymorphisms (SNPs; Roden et al, 2009;Hancock-Hanser et al, 2013;Hurtado et al, 2016). With many alleles at each locus, nuclear microsatellites can be employed for applications such as individual and familial genotyping (Selkoe and Toonen, 2006).…”

“…For example, in addition to DNA sequencing HTS techniques, RNA sequencing (RNASeq) can be employed to quantify gene expression (in lieu of lower throughput techniques such as quantitative PCR) and conduct functional genomics studies. Although to date HTS has been more widely used in other wildlife taxa (e.g., mammals and fish; Shafer et al, 2015;Cammen et al, 2016), in marine turtles HTS approaches have been used to construct the complete green turtle reference genome (Wang et al, 2013), generate mitogenomes of all seven marine turtle species for phylogenetic analyses (Duchêne et al, 2012), and discover SNPs for green, hawksbill and loggerhead turtles (Hancock-Hanser et al, 2013;Komoroske et al, 2016). While HTS approaches effectively eliminate bottlenecks in data production, challenges in data quality and interpretation can remain, particularly for wildlife species with limited genomic resources.…”

Surface properties of a laser-treated biopolymer

“…Nanostructured materials find different applications, e.g., in DNA and protein sequencing 2 , in the creation of a suitable synthetic environment for cell growth 3 or in the solar-cell technology. 4 Self-organized structures are prepared with a bottom-up method.…”