Thursday, December 23, 2010

Comparison of various DNA-Seq library prep methods

Adey et al. (in the laboratory of Jay Shendure, University of Washington) recently published a methods paper characterizing various library prep technologies for high-throughput DNA sequencing, including Epicentre’s Nextera™ technology. The publication highlights recent advances in DNA library preparation for next-generation sequencing, in order to overcome the bottleneck posed by earlier methods, i.e., labor, time, and lack of automation.

With Nextera technology, it is now possible to prepare literally hundreds of libraries in a day.  With respect to bias, the authors state: 
Comparison to conventional methods of library preparation, relying on mechanical or endonuclease fragmentation, finds that although transposase-catalyzed adaptor insertion demonstrates a slightly greater insertion bias, this has little impact at the level of genomic coverage, and is offset by large advantages with respect to speed, simplicity, and low input requirements. 
As described in the paper, the Nextera system is highly versatile, and can be adapted to multiple applications. These include library preparation from as little as 10 pg DNA, exome capture, PCR-free and colony PCR library preparation, and sample prep automation.

ResearchBlogging.orgAdey, A. et al. (2010). Rapid, low-input, low-bias construction of shotgun fragment libraries by high-density in vitro transposition Genome Biology, 11 (12) DOI: 10.1186/gb-2010-11-12-r119

Monday, December 20, 2010

Quantitation of input DNA and Nextera™ fragment size distribution

When preparing Illumina-compatible Nextera libraries, it is critical to accurately quantify the input DNA amount. Variation in input DNA can affect the molecular weight (MW) distribution of libraries. The Bioanalyzer traces below show size distribution as a function of input DNA amount (human genomic DNA). As the traces show, the MW distribution increases (larger fragments) with the input DNA amount. Please note that the degree of variance will depend on a number of factors, including sample type and purity. For accurate DNA quantitation, any of the following methods can be used: qPCR, Qubit® fluorimetry, or NanoDrop™ analysis.

If a stringent size distribution is needed, there are a number of methods that can be used to size-select for the required fragment size, including AMPure® XP (for removing small fragments), Caliper LabChip® XT (for very narrow size selection), or gel extraction.

Fragment size and amount of input DNA (click to enlarge figure). Varying amounts of human genomic DNA were tagmented with Illumina-compatible Nextera™ Enzyme Mix. Tagmentation was performed using the HMW buffer, followed by nine cycles of PCR and DNA clean-up (without size selection). Red: 25 ng; Blue: 50 ng; Green: 100 ng; Aqua: 150 ng.

Wednesday, December 15, 2010

BAC libraries enable phylogenetic analysis in Japanese pear

With the rapid expansion of next-generation sequencing, there has been speculation that the backbone technology of older genomic sequencing, large-insert cloning (e.g., using bacterial artificial chromosomes [BACs]) would become less important. However, in many laboratories, BAC libraries still play a role in sequencing and gene expression studies.

In a recent study, Okada et al. screened a library created in the CopyControl™ BAC Cloning Kit (Hind III cloning-ready vector) to study the mechanics of self-incompatibility (inhibiting self-fertilization) in the Japanese pear (Pyrus pyrifolia). The work focused on a DNA contig that contained a 649-kb region around the S-RNase genes. After creation of the BAC library, the selected clones were sequenced using standard ABI BigDye 3.1 sequencing techniques and “old-fashioned” chromosome walking. However, BAC library screening services are increasing turning to next-generation sequencing technologies, due to the ease of rapidly generating large amounts of sequence data with better depth and coverage than traditional Sanger sequencing.

ResearchBlogging.orgOkada, K. et al. (2010). Related polymorphic F-box protein genes between haplotypes clustering in the BAC contig sequences around the S-RNase of Japanese pear Journal of Experimental Botany DOI: 10.1093/jxb/erq381

Friday, December 10, 2010

Microsatellite loci for Symbiodinium A3 Identified using next-generation sequencing

Microsatellites, or simple sequence repeats (SSRs), are molecular markers that can be readily investigated for population genetic studies. Microsatellites contain tandem repeats of 1-6 bases and are usually highly polymorphic, displaying a large number of alleles. The high degree of polymorphism makes microsatellites an ideal tool for studying gene-flow.

A recent study by Pinzon et al. developed ten polymorphic microsatellite loci for a common algae (Symbiodinium fitti, type A3) to study coral-algal symbioses. For this study, genomic DNA from three cultured strains of S. fitti were extracted and purified. Two different methods were employed to identify microsatellite loci with di-, tri-, and tetranucleotide motifs: i) Roche 454 sequencing; and ii) standard clone library (TA cloning) amplified and cycle sequenced using the ABI’s Big Dye Terminator Kit. For the Roche 454 sequencing, libraries were prepared from only 50 ng of double-stranded DNA using the Nextera™ DNA Sample Prep Kit (FLX Titanium-compatible), and sequenced on the 454 GS-FLX sequencer. The sequencing results helped the authors identify three to eight alleles for each haploid locus, with <95% of the samples possessing a single, symbiont, multilocus genotype (MLG).

The study demonstrates the utility of next-generation sequencing (NGS), especially with limited amounts of DNA. As shown in this study, NGS can be used to identify population genetic markers, which can help scientists better understand intraspecific and interspecific gene flow and population genetic structure.

ResearchBlogging.orgPinzón, J. et al. (2010). Microsatellite loci for Symbiodinium A3 (S. fitti) a common algal symbiont among Caribbean Acropora (stony corals) and Indo-Pacific giant clams (Tridacna) Conservation Genetics Resources, 3 (1), 45-47 DOI: 10.1007/s12686-010-9283-5

Thursday, December 2, 2010

New small-RNA-Seq library prep kit

Epicentre recently introduced the ScriptMiner™ Small RNA-Seq Library Preparation Kit (SinglePlex; Illumina-compatible). The kit produces nonbarcoded (singleplex) sequencing libraries from miRNA and, optionally, small 5'-capped RNA and 5'-triphosphorylated RNA. The ScriptMiner process includes a unique enzymatic procedure that removes excess 3' adaptor oligo, greatly reducing the amount of adaptor-dimer products in the sequencing library.

Small-RNA coverage from ScriptMiner™ and conventional libraries (click to enlarge figure). Small-RNA libraries were prepared using ScriptMiner (left panel) and conventional (right panel) methods and sequenced on an Illumina® GAIIx sequencer.  Both libraries produced similar numbers of aligned reads; however, the ScriptMiner library had a higher proportion of mature miRNA sequences.