Microsatellite markers from the Ion Torrent: a multi‐species contrast to 454 shotgun sequencing

Abstract

The development and screening of microsatellite markers has been accelerated by next 35 generation sequencing (NGS) technology and in particular GS-FLX pyro-sequencing (454). 36More recent platforms such as the PGM semi-conductor sequencer (Ion Torrent) offer 37 potential benefits such as dramatic reductions in cost, but to date have not been well utilised. 38Here we critically compare the advantages and disadvantages of microsatellite development 39 using PGM semi-conductor sequencing and GS-FLX pyro-sequencing for two gymnosperm 40(a conifer and a cycad) and one angiosperm species. We show that these NGS platforms differ 41 in the quantity of returned sequence data, unique microsatellite data and primer design 42 opportunities, mostly consistent with the differences in read length. The strength of the PGM 43 lies in the large amount of data generated at a comparatively lower cost and time. The strength 44 of GS-FLX lies in the return of longer average length sequences and therefore greater 45 flexibility in producing markers with variable product length, due to longer flanking regions, 46 which is ideal for capillary multi-plexing. These differences need to be considered when 47 choosing a NGS method for microsatellite discovery. However, the ongoing improvement in 48

The report of my death was an exaggeration: A review for researchers using microsatellites in the 21st century

“…Studies so far have largely focused microsatellite discovery efforts on the Roche 454 (454 Life Sciences, a Roche Company, Branford, Connecticut, USA) and Illumina (Illumina, San Diego, California) platforms (Jennings et al, 2011; Zalapa et al, 2012), although Pacific Biosciences (PacBio, Menlo Park, California, USA) (Grohme et al, 2013; Wei et al, 2014) and Ion Torrent (Thermo Fisher Scientific, Waltham, Massachusetts, USA) (Huey et al, 2013; Kameyama and Hirao, 2014) have also been used. Because read length greatly affects the ability to discover microsatellite markers, as longer reads will more likely include the flanking regions needed for primer design (Lepais and Bacles, 2011; Schoebel et al, 2013; Elliott et al, 2014), the 454 sequencing platform was used extensively for microsatellite development (Castoe et al, 2010). On a per-megabase basis, however, 454 is less cost-effective than Illumina (Glenn, 2011; ).…”

“…Gilmore et al (2013) estimated the time and cost of using Illumina data to produce markers from eight samples to be approximately 20 h of laboratory work for sample preparation and approximately US$51 per sample. Several recent studies have justified the use of other platforms, mainly Ion Torrent (Elliott et al, 2014) and PacBio (Wei et al, 2014). In a comparison of the utility of 454 and Ion Torrent, Elliott et al (2014) found the Ion Torrent recovered shorter microsatellite repeats (due to shorter reads), but more markers were discovered at a lower cost and more quickly than with 454.…”

QDD version 3.1: a user‐friendly computer program for microsatellite selection and primer design revisited: experimental validation of variables determining genotyping success rate

“…Although there is no theoretical reason to exclude these types of sequences for QDD3 analyses, there are two potential problems. The typical read length of 100-250 bp of these platforms (especially for earlier versions) would provide only short markersmaking multiplexing less efficientand the chance of finding a sufficient flanking region of both sides of the microsatellites is lower than for long reads (Elliott et al 2014). Furthermore, it is more likely that primers are designed close to the target microsatellites and thus increasing the risk of PCR failure.…”

“…; Elliott et al . ). As the fastq is the standard output format of these last two technologies, QDD3 can also accept this format as an input to facilitate the use of the pipeline.…”

“…Thirdly, QDD was originally designed to analyse 454 sequences, as this was the only available NGS platform that produced reads long enough for marker design. Recently, both Ion Torrent and paired-end Illumina sequences have been shown to be suitable for microsatellite marker design (Castoe et al 2012;Elliott et al 2014). As the fastq is the standard output format of these last two technologies, QDD3 can also accept this format as an input to facilitate the use of the pipeline.…”