Multiplexed microsatellite recovery using massively parallel sequencing

Abstract

Conservation and management of natural populations requires accurate and inexpensive genotyping methods. Traditional microsatellite, or simple sequence repeat (SSR), marker analysis remains a popular genotyping method because of the comparatively low cost of marker development, ease of analysis and high power of genotype discrimination. With the availability of massively parallel sequencing (MPS), it is now possible to sequence microsatellite-enriched genomic libraries in multiplex pools. To test this approach, we prepared seven microsatellite-enriched, barcoded genomic libraries from diverse taxa (two conifer trees, five birds) and sequenced these on one lane of the Illumina Genome Analyzer using paired-end 80-bp reads. In this experiment, we screened 6.1 million sequences and identified 356,958 unique microreads that contained di- or trinucleotide microsatellites. Examination of four species shows that our conversion rate from raw sequences to polymorphic markers compares favourably to Sanger- and 454-based methods. The advantage of multiplexed MPS is that the staggering capacity of modern microread sequencing is spread across many libraries; this reduces sample preparation and sequencing costs to less than $400 (USD) per species. This price is sufficiently low that microsatellite libraries could be prepared and sequenced for all 1373 organisms listed as 'threatened' and 'endangered' in the United States for under $0.5 M (USD).

The Parasitic Plant Genome Project: New Tools for Understanding the Biology ofOrobancheandStriga

“…High-copy chloroplast genomes can often be fully recovered and can be used to create sequencing primers ( Straub et al, 2011 ). Another application for low-coverage WGS includes the identifi cation of microsatellite loci ( Jennings et al, 2011 ;Castoe et al, 2012 ). The discovery of low-copy nuclear loci, on the other hand, can be more diffi cult as very-low-coverage genome skimming may produce only small fragments (e.g., ~400-500 bp on the Roche 454) when reads cannot be assembled into contigs.…”

Microsatellite Marker Discovery using Single Molecule Real-Time Circular Consensus Sequencing on the Pacific Biosciences RS

“…This is equivalent to a(combined) locus to primer conversion of 90%. This yield is higher than the values obtained from 454 Titanium or Illumina shotgun sequencing, which usually show conversion of 40%60% of the loci due to read length constraints and depending on stringency of analysis (2, 4, 5, 11). …”

“…Several price calculations for different PacBio sequencing chemistry combinations can be found in the recent study by Koren et al (18). This puts the cost of microsatellite discovery using PacBio sequencing between that of 454 and Illumina sequencing (4). Our approach provides a useful alternative when cost reduction using multiplexing is not practicable, and microsatellite array length is to be determined directly from the data for prioritized locus testing .…”

“…The first high-throughput attempts to identify microsatellite markers were performed using 454 pyrosequencing of genomic shotgun libraries or microsatellite enriched libraries (13). Approaches using ultra-short read sequencing for microsatellite identification have also been explored (4, 5). Typically, these cloning-free approaches are performed at low genome coverage, employing relatively short sequence reads and often recovering small fractions of the target genome.…”

Development of genomic microsatellites in Gleditsia triacanthos (Fabaceae) using Illumina sequencing

“…Low-coverage whole genome sequencing (Jennings et al, 2011) was used to produce an initial set of genomic resources for 10 hardwood tree species, including honey locust (Staton et al, in prep.). Illumina libraries were created from sonicated genomic DNA extracted from leaflets of one individual (seed parent of 88 single-tree progeny, Appendix 1) of G. triacanthos per the manufacturer’s protocol (QIAGEN DNeasy96 Plant Kit; QIAGEN, Hilden, Germany).…”