A scalable, fully automated process for construction of sequence-ready barcoded libraries for 454

Lennon, Niall J.; Lintner, Robert E.; Anderson, Scott; Álvarez, Pablo; Barry, Andrew; Brockman, William W.; Daza, Riza M.; Erlich, Rachel; Giannoukos, Georgia; Green, Lisa; Hollinger, Andrew; Hoover, Cindi A.; Jaffe, David B.; Juhn, Frank; McCarthy, Danielle; Perrin, Danielle; Ponchner, Karen; Powers, Taryn L; Rizzolo, Kamran; Robbins, Dana; Ryan, Elizabeth; Russ, Carsten; Sparrow, Todd; Stalker, John; Steelman, Scott; Weiand, Michael; Zimmer, Andrew; Henn, Matthew R.; Nusbaum, Chad; Nicol, Robert

doi:10.1186/gb-2010-11-2-r15

Cited by 92 publications

(101 citation statements)

References 23 publications

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“…PCR products were quantified with the Quant-It DNA assay kit (Invitrogen), normalized, sheared, and barcoded by sample. Samples were pooled and sequenced on a 454/Roche high-throughput genome sequencer as previously described (28,40), with a target average sequence coverage of 250-fold for each residue in the DENV genome per sample, allowing for the capture of variants that are present at Ͼ1% of the viral population.…”

Section: Methodsmentioning

confidence: 99%

Genome-Wide Patterns of Intrahuman Dengue Virus Diversity Reveal Associations with Viral Phylogenetic Clade and Interhost Diversity

Parameswaran

Charlebois

Téllez

et al. 2012

J Virol

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Analogous to observations in RNA viruses such as human immunodeficiency virus, genetic variation associated with intrahost dengue virus (DENV) populations has been postulated to influence viral fitness and disease pathogenesis. Previous attempts to investigate intrahost genetic variation in DENV characterized only a few viral genes or a limited number of full-length genomes. We developed a whole-genome amplification approach coupled with deep sequencing to capture intrahost diversity across the entire coding region of DENV-2. Using this approach, we sequenced DENV-2 genomes from the serum of 22 Nicaraguan individuals with secondary DENV infection and captured ϳ75% of the DENV genome in each sample (range, 40 to 98%). We identified and quantified variants using a highly sensitive and specific method and determined that the extent of diversity was considerably lower than previous estimates. Significant differences in intrahost diversity were detected between genes and also between antigenically distinct domains of the Envelope gene. Interestingly, a strong association was discerned between the extent of intrahost diversity in a few genes and viral clade identity. Additionally, the abundance of viral variants within a host, as well as the impact of viral mutations on amino acid encoding and predicted protein function, determined whether intrahost variants were observed at the interhost level in circulating Nicaraguan DENV-2 populations, strongly suggestive of purifying selection across transmission events. Our data illustrate the value of high-coverage genome-wide analysis of intrahost diversity for high-resolution mapping of the relationship between intrahost diversity and clinical, epidemiological, and virological parameters of viral infection. N early three billion people worldwide are at risk for infection with dengue virus (DENV), a Flavivirus transmitted by the mosquitoes Aedes aegypti and A. albopictus (for reviews, see references 24 and 25). DENV is an RNA virus with a single-stranded, nonsegmented RNA genome ϳ10.7 kb in length, which encodes three structural proteins (capsid [C], premembrane/membrane [prM/M], and envelope ]E]) and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). There are four closely related serotypes of DENV (DENV-1 to DENV-4) that can cause asymptomatic infections or clinical illnesses ranging from the self-limiting but debilitating dengue fever to the lifethreatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), which are characterized by vascular leakage (76). Even though several factors, such as viral genetics, preexisting heterotypic immunity (i.e., immunity to a different serotype due to a prior infection), the sequence of infection with distinct serotypes, and host genetics appear to influence disease severity (23,35,53,54,58,61,69), the precise causes for progression to severe disease remain unknown.The four DENV serotypes share limited identity, with 25 to 40% variability observed between serotypes at the amino acid level (30, 68)....

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Section: Methodsmentioning

confidence: 99%

Genome-Wide Patterns of Intrahuman Dengue Virus Diversity Reveal Associations with Viral Phylogenetic Clade and Interhost Diversity

Parameswaran

Charlebois

Téllez

et al. 2012

J Virol

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“…Such methodology has proven very efficient for outlier type analyses (see below) but may also be applicable in other kinds of population genetic studies. Tagging of samples, which is necessary for investigating genetic variation on an individual level, will be facilitated by the use of a recently launched highly automated procedure (Lennon et al, 2010). The trade off between sequence depth of individual samples and number of samples sequenced also needs to be considered, as the calculations of allele frequencies and detection of low-frequency SNPs will be severely hampered if there is insufficient coverage of individual SNPs.…”

Section: Nucleotide Variation Profilingmentioning

confidence: 99%

Applications of next generation sequencing in molecular ecology of non-model organisms

2010

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As most biologists are probably aware, technological advances in molecular biology during the last few years have opened up possibilities to rapidly generate large-scale sequencing data from non-model organisms at a reasonable cost. In an era when virtually any study organism can 'go genomic', it is worthwhile to review how this may impact molecular ecology. The first studies to put the next generation sequencing (NGS) to the test in ecologically well-characterized species without previous genome information were published in 2007 and the beginning of 2008. Since then several studies have followed in their footsteps, and a large number are undoubtedly under way. This review focuses on how NGS has been, and can be, applied to ecological, population genetic and conservation genetic studies of non-model species, in which there is no (or very limited) genomic resources. Our aim is to draw attention to the various possibilities that are opening up using the new technologies, but we also highlight some of the pitfalls and drawbacks with these methods. We will try to provide a snapshot of the current state of the art for this rapidly advancing and expanding field of research and give some likely directions for future developments.

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“…16S rRNA V1-V3 amplicon libraries were prepared from sample DNA using AccuPrime HF Taq (Invitrogen, 12346-086) and universal primers flanking variable regions V1 (27F, 59-AGAG TTTGATCCTGGCTCAG-39) and V3 (534R, 59-ATTACCGCGGCT GCTGG-39). For each sample, the universal primers were tagged with unique sequences (''barcodes'') to allow for multiplexing/ demultiplexing (Lennon et al 2010). For ITS1 amplicon library preparation, universal primers flanking the ITS1 region-adapter + 18SF (59-CCTATCCCCTGTGTGCCTTGGCAGTCTCAGGTAAAAG TCGTAACAAGGTTTC-39) and 5.8S-1R + barcode (59-GTTCAAAG AYTCGATGATTCAC-39)-were used to amplify a select number of genomic DNA extractions.…”

Section: Preparation Of Samples For 454 Sequencingmentioning

confidence: 99%

The altered landscape of the human skin microbiome in patients with primary immunodeficiencies

et al. 2013

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While landmark studies have shown that microbiota activate and educate host immunity, how immune systems shape microbiomes and contribute to disease is incompletely characterized. Primary immunodeficiency (PID) patients suffer recurrent microbial infections, providing a unique opportunity to address this issue. To investigate the potential influence of host immunity on the skin microbiome, we examined skin microbiomes in patients with rare monogenic PIDs: hyper-IgE (STAT3-deficient), Wiskott-Aldrich, and dedicator of cytokinesis 8 syndromes. While specific immunologic defects differ, a shared hallmark is atopic dermatitis (AD)-like eczema. We compared bacterial and fungal skin microbiomes (41 PID, 13 AD, 49 healthy controls) at four clinically relevant sites representing the major skin microenvironments. PID skin displayed increased ecological permissiveness with altered population structures, decreased site specificity and temporal stability, and colonization with microbial species not observed in controls, including Clostridium species and Serratia marcescens. Elevated fungal diversity and increased representation of opportunistic fungi (Candida, Aspergillus) supported increased PID skin permissiveness, suggesting that skin may serve as a reservoir for the recurrent fungal infections observed in these patients. The overarching theme of increased ecological permissiveness in PID skin was counterbalanced by the maintenance of a phylum barrier in which colonization remained restricted to typical human-associated phyla. Clinical parameters, including markers of disease severity, were positively correlated with prevalence of Staphylococcus, Corynebacterium, and other less abundant taxa. This study examines differences in microbial colonization and community stability in PID skin and informs our understanding of host-microbiome interactions, suggesting a bidirectional dialogue between skin commensals and the host organism.

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A scalable, fully automated process for construction of sequence-ready barcoded libraries for 454

Abstract: An automated method for constructing libraries for 454 sequencing significantly reduces the cost and time required.

Cited by 92 publications

References 23 publications

Genome-Wide Patterns of Intrahuman Dengue Virus Diversity Reveal Associations with Viral Phylogenetic Clade and Interhost Diversity

Genome-Wide Patterns of Intrahuman Dengue Virus Diversity Reveal Associations with Viral Phylogenetic Clade and Interhost Diversity

Applications of next generation sequencing in molecular ecology of non-model organisms

The altered landscape of the human skin microbiome in patients with primary immunodeficiencies

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