Wednesday, 5 October 2011

What's new for 'next generation sequencing' in PubMed

1.Analysis of 16S rRNA Amplicon Sequencing Options on the Roche/454 Next-Generation Titanium Sequencing Platform.
Tamaki H, Wright CL, Li X, Lin Q, Hwang C, Wang S, Thimmapuram J, Kamagata Y, Liu WT.
PLoS One. 2011;6(9):e25263. Epub 2011 Sep 23.
PMID: 21966473 [PubMed - in process]



16S rRNA gene pyrosequencing approach has revolutionized studies in microbial ecology. While primer selection and short read length can affect the resulting microbial community profile, little is known about the influence of pyrosequencing methods on the sequencing throughput and the outcome of microbial community analyses. The aim of this study is to compare differences in output, ease, and cost among three different amplicon pyrosequencing methods for the Roche/454 Titanium platform


The following three pyrosequencing methods for 16S rRNA genes were selected in this study: Method-1 (standard method) is the recommended method for bi-directional sequencing using the LIB-A kit; Method-2 is a new option designed in this study for unidirectional sequencing with the LIB-A kit; and Method-3 uses the LIB-L kit for unidirectional sequencing. In our comparison among these three methods using 10 different environmental samples, Method-2 and Method-3 produced 1.5-1.6 times more useable reads than the standard method (Method-1), after quality-based trimming, and did not compromise the outcome of microbial community analyses. Specifically, Method-3 is the most cost-effective unidirectional amplicon sequencing method as it provided the most reads and required the least effort in consumables management.


Our findings clearly demonstrated that alternative pyrosequencing methods for 16S rRNA genes could drastically affect sequencing output (e.g. number of reads before and after trimming) but have little effect on the outcomes of microbial community analysis. This finding is important for both researchers and sequencing facilities utilizing 16S rRNA gene pyrosequencing for microbial ecological studies.

[PubMed - in process]

PMCID: PMC3179495

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2.Insight into the heterogeneity of breast cancer through next-generation sequencing.
Russnes HG, Navin N, Hicks J, Borresen-Dale AL.
J Clin Invest. 2011 Oct 3;121(10):3810-8. doi: 10.1172/JCI57088. Epub 2011 Oct 3.
PMID: 21965338 [PubMed - in process]


Rapid and sophisticated improvements in molecular analysis have allowed us to sequence whole human genomes as well as cancer genomes, and the findings suggest that we may be approaching the ability to individualize the diagnosis and treatment of cancer. This paradigmatic shift in approach will require clinicians and researchers to overcome several challenges including the huge spectrum of tumor types within a given cancer, as well as the cell-to-cell variations observed within tumors. This review discusses how next-generation sequencing of breast cancer genomes already reveals insight into tumor heterogeneity and how it can contribute to future breast cancer classification and management.

[PubMed - in process]

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3. Using next generation sequencing to identify yellow fever virus in Uganda.
McMullan LK, Frace M, Sammons SA, Shoemaker T, Balinandi S, Wamala JF, Lutwama JJ, Downing RG, Stroeher U, Macneil A, Nichol ST.
Virology. 2011 Sep 30. [Epub ahead of print]


In October and November 2010, hospitals in northern Uganda reported patients with suspected hemorrhagic fevers. Initial tests for Ebola viruses, Marburg virus, Rift Valley fever virus, and Crimean Congo hemorrhagic fever virus were negative. Unbiased PCR amplification of total RNA extracted directly from patient sera and next generation sequencing resulted in detection of yellow fever virus and generation of 98% of the virus genome sequence. This finding demonstrated the utility of next generation sequencing and a metagenomic approach to identify an etiological agent and direct the response to a disease outbreak.

Copyright © 2011. Published by Elsevier Inc.

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PMID: 21962764 [PubMed - as supplied by publisher]
4.A blueprint for advancing genetics-based cancer therapy.
Sellers WR.
Cell. 2011 Sep 30;147(1):26-31.
PMID: 21962504 [PubMed - in process]
5.Unraveling the Chinese hamster ovary cell line transcriptome by next-generation sequencing.
Becker J, Hackl M, Rupp O, Jakobi T, Schneider J, Szczepanowski R, Bekel T, Borth N, Goesmann A, Grillari J, Kaltschmidt C, Noll T, PĆ¼hler A, Tauch A, Brinkrolf K.
J Biotechnol. 2011 Sep 17. [Epub ahead of print]
PMID: 21945585 [PubMed - as supplied by publisher]


The pyrosequencing technology from 454 Life Sciences and a novel assembly approach for cDNA sequences with the Newbler Assembler were used to achieve a major step forward to unravel the transcriptome of Chinese hamster ovary (CHO) cells. Normalized cDNA libraries originating from several cell lines and diverse culture conditions were sequenced and the resulting 1.84 million reads were assembled into 32,801 contiguous sequences, 29,184 isotigs, and 24,576 isogroups. A taxonomic classification of the isotigs showed that more than 70% of the assembled data is most similar to the transcriptome of Mus musculus, with most of the remaining isotigs being homologous to DNA sequences from Rattus norvegicus. Mapping of the CHO cell line contigs to the mouse transcriptome demonstrated that 9124 mouse transcripts, representing 6701 genes, are covered by more than 95% of their sequence length. Metabolic pathways of the central carbohydrate metabolism and biosynthesis routes of sugars used for protein N-glycosylation were reconstructed from the transcriptome data. All relevant genes representing major steps in the N-glycosylation pathway of CHO cells were detected. The present manuscript represents a data set of assembled and annotated genes for CHO cells that can now be used for a detailed analysis of the molecular functioning of CHO cell lines.

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