Comparison of commercially available target enrichment methods for next-generation sequencing.

Isolating high-priority segments of genomes greatly enhances the efficiency of next-generation sequencing (NGS) by allowing researchers to focus on their regions of interest. For the 2010-11 DNA Sequencing Research Group (DSRG) study, we compared outcomes from two leading companies, Agilent Technologies (Santa Clara, CA, USA) and Roche NimbleGen (Madison, WI, USA), which offer custom-targeted genomic enrichment methods. Both companies were provided with the same genomic sample and challenged to capture identical genomic locations for DNA NGS.

Sequencing of the Dutch elm disease fungus genome using the Roche/454 GS-FLX Titanium System in a comparison of multiple genomics core facilities.

As part of the DNA Sequencing Research Group of the Association of Biomolecular Resource Facilities, we have tested the reproducibility of the Roche/454 GS-FLX Titanium System at five core facilities. Experience with the Roche/454 system ranged from <10 to >340 sequencing runs performed. All participating sites were supplied with an aliquot of a common DNA preparation and were requested to conduct sequencing at a common loading condition.

Identification of optimal protocols for sequencing difficult templates: results of the 2008 ABRF DNA Sequencing Research Group difficult template study 2008.

The 2008 ABRF DNA Sequencing Research Group (DSRG) difficult template sequencing study was designed to identify a general set of guidelines that would constitute the best approaches for sequencing difficult templates. This was a continuation of previous DSRG difficult template studies performed in 1996, 1997, and 2003. The distinguishing factors in the present study were the number of DNA templates used, the number of different types of difficult regions tested, and the inclusion of a follow-up phase of the study to identify optimal protocols for each type of difficult template.

Strategies for genotyping: Effectiveness of tailing primers to increase accuracy in short tandem repeat determinations.

A problem associated with automated analysis of fluorescently labeled fragments separated by slab gel or capillary electrophoresis is the doublet peak formed when Taq DNA Polymerase adds a nontemplated nucleotide (generally an adenosine) to the 3' end of the product.This nontemplated addition (plus A) is primarily dependent on the 5' sequence of the reverse primer and, to a lesser extent, polymerase chain reaction (PCR) conditions. Primers may amplify the true product, the plus A product or a doublet product comprised of both.

10-nmol oligonucleotide synthesis for the ABI model 394 DNA synthesizer.

Sequencing and gene contig assembly generally involve primer walking, in which an oligonucleotide primer is used once and then discarded. Because the smallest commonly available scale for commercial oligonucleotide synthesizers is the 40-nmol scale, a large excess of product is produced. This results in reagent waste, excess cost to the researcher, and increased production time.