Thursday, December 22, 2011

Large-insert cloning aids study of dinoflagellate species

Large-insert cloning products have long been a mainstay of Epicentre's product line and are critical for the study of gene expression and interactions. Recently, Jaeckisch et al. described making three kinds of libraries (cDNA, fosmid, and BAC) for characterizing the marine dinoflagellate Alexandrium ostenfeldii.

Many dinoflagellate species are notorious for the toxins they produce, as well as ecological and human health consequences associated with harmful algal blooms (HABs). One way to study these otherwise toxic compounds is to build their synthesis genes into a large-insert-capable, low-copy cloning vector, transform into a suitable host cell, and then perform the desired studies in such a way that the genes will not have a negative affect on the host. The genes for the toxins cited in the publication (macrocyclic imine toxins, described as spirolides), were inserted into the pCC1FOS vector and transfected into the TransforMAX™ EPI300 host strain for further study. Further, genomic DNA from A. ostefendii was prepared for cloning into the pIndigoBAC-5 HindIII Cloning-Ready vector and transformation into the TransforMAX EC100 host.

A total of 384 BAC clones were obtained with insert sizes ranging from 50 to 150 kb, which provided sufficient coverage to allow elucidation of the whole genome sequence and some comparative sequence data with the marine dinoflagellate H. triquetra. The authors used the sequence information obtained from the BAC and CopyControl fosmid libraries to investigate spliced leader (SL) trans-splicing and mRNA transposition mechanisms. They characterized the genome using selected clones, using a combination of transcriptomic data and random genomic clones. Examination of SL sequences revealed similar features as in other dinoflagellates, including other Alexandrium species. SL sequences in decay indicate frequent retro-transposition of mRNA species. The transposition observed in these genes probably contributes to overall genome complexity by generating additional gene copies.
The authors conclude:
The genomic sequence survey (GSS) represents the largest genomic data set of a dinoflagellate to date...The large portion of repetitive sequences and the organization within the genome is in agreement with several other studies on dinoflagellates using different approaches. It remains to be determined whether this unusual composition is directly correlated to the exceptionally [sic] genome organization of dinoflagellates with a low amount of histones and histone-like proteins.
ResearchBlogging.orgJaeckisch, N. et al. (2011). Comparative Genomic and Transcriptomic Characterization of the Toxigenic Marine Dinoflagellate Alexandrium ostenfeldii PLoS ONE, 6 (12) DOI: 10.1371/journal.pone.0028012

Monday, December 12, 2011

What's new in the ScriptSeq v2 kit?

Recently, Epicentre launched the ScriptSeq™ v2 RNA-Seq Kit for preparing directional, ligation-free RNA-Seq libraries in 4 hours. This kit offers several advantages over the previous version:
  • Transcript coverage is improved and GC bias is reduced by an improved terminal-tagging oligo (TTO).
  • Less input RNA required: Libaries can be prepared from as little as 500 pg of rRNA-depleted or poly(A)-enriched RNA.
  • A streamlined protocol and premixed reagents make the kit easier to use and require fewer pipetting steps.
  • A signifcantly lower price compared to the original kit.
The original ScriptSeq Kit will be available for a transition period, to allow existing customers to finish important projects. We expect customers will find that the new ScriptSeq v2 Kit provides better performance at a lower price.

Friday, December 9, 2011

Role of Piwi proteins in mammalian transposon silencing

In a recent Nature publication, Reuter et al. report on the study of certain small RNAs that affect the fertility of male mice. Piwi-interacting RNAs (piRNAs) act together with Piwi proteins Mili (also known as Piwil2) and Miwi (also known as Piwil4) in a genome defense mechanism that initiates transposon silencing via DNA methylation in the mouse male embryonic germ line. This silencing depends on the participation of the Piwi proteins in a slicer-dependent piRNA amplification pathway and is essential for male fertility.

The researchers cite the use of Epicentre’s Ribo-Zero™ Kit (Human/Mouse/Rat) to remove interfering ribosomal RNA from the experimental matrix, allowing closer study of the interactions between piRNAs. They also used the ScriptSeq™ mRNA-Seq Kit for an unusual application: sequencing small RNAs. Under normal circumstances, small RNAs <50-60 nucleotides are better suited for Epicentre's ScriptMiner™ Small RNA-Seq Kit, due to potential issues with the ScriptSeq method at the 5’ end of small RNAs. These results demonstrate that the ScriptSeq Kit has the ability to prepare RNA-Seq libraries from RNA types other than mRNA, including small RNAs.

ResearchBlogging.orgReuter, M. et al. (2011). Miwi catalysis is required for piRNA amplification-independent LINE1 transposon silencing Nature, 480 (7376), 264-267 DOI: 10.1038/nature10672

Friday, December 2, 2011

Transposome-based RNA-Seq library construction from low input amounts of RNA

A collaborative effort between Epicentre and the HudsonAlpha Institute for Biotechnology resulted in the development of two novel transposon-based methods for RNA-Seq library preparation. The technique, called Tn-RNA-Seq, can use double-stranded cDNA created from rRNA-depleted RNA to prepare an Illumina® sequencing library using only two enzymatic reactions. The researchers generated high-quality RNA-Seq libraries from as little as 10 pg of mRNA (~1 ng of total RNA) with this approach.

They also present a strand-specific RNA-Seq library construction protocol that combines transposon-based library construction with uracil DNA glycosylase and Endonuclease VIII to specifically degrade the second strand constructed during cDNA synthesis. These directional RNA-Seq libraries maintained the same quality as the nondirectional libraries, while showing a high degree of strand specificity (99.5% of reads mapped to the expected genomic strand).

A key benefit of the Tn-RNA-Seq technique is the ability to use extremely low amounts of RNA to prepare high-quality libraries. All six libraries generated using 10 ng to 10 pg of mRNA had at least 72% of aligned reads map to known transcripts, while the library made from 1 pg of mRNA had 62% of aligned reads map to known transcripts. Library complexity was found to be high for all libraries except for the library constructed with 1 pg of mRNA. In general, Tn-RNASeq libraries made with 10 pg or more of mRNA (about 50 cell equivalents) exhibited consistent quality measures. For all libraries except for the library made with 1 pg of mRNA, the rank correlations remained very high (>0.96) indicating highly consistent and reproducible library formation. The directional Tn-RNA-Seq libraries retained the same level of “strandedness” during sequencing compared to libraries made using standard adaptor-ligation methods.

The authors concluded that high-quality RNA-Seq libraries can be constructed efficiently from low input amounts of RNA using the Tn-RNA-Seq methods, and that the procedure is suitable for high-throughput or automated workflows.

ResearchBlogging.orgGertz, J. et al. (2011). Transposase mediated construction of RNA-seq libraries Genome Research DOI: 10.1101/gr.127373.111