Next-generation DNA Sequencing Platforms Research Articles

Concurrent PTEN and PDGFRB Alterations Characterize Storiform Collagenoma.

Storiform collagenoma is a rare mesenchymal skin tumor that is composed of thickened collagen bundles arranged in a characteristic storiform pattern with a relatively hypocellular CD34-positive spindle cell component. Storiform collagenoma is most often sporadic, but multiple lesions can occur in Cowden syndrome, which is characterized by germline alterations in PTEN (phosphatase and tensin homolog) on chromosome 10. Here, we investigated the molecular pathogenesis of storiform collagenoma using a targeted next-generation DNA sequencing platform, including 5 sporadic cases and one case associated with Cowden syndrome. Recurrent PTEN alterations were identified in all cases, with biallelic PTEN inactivation observed in the case associated with Cowden syndrome and one sporadic case. Unexpectedly, we also identified recurrent activating mutations in the platelet-derived growth factor receptor beta (PDGFRB) gene. This included a missense substitution in the D5 Ig-like domain of PDGFRB in the Cowden syndrome-associated case. In addition, we report missense alterations in the juxtamembrane domain of PDGFRB in 4 of 5 (80%) sporadic cases, including mutations that have been previously described in sporadic myofibroma and myopericytoma. Therefore, we confirm the neoplastic nature of storiform collagenoma, we expand the spectrum of reported PDGFRB alterations in mesenchymal tumors and we suggest a possible collaborative role for PTEN and PDGFRB in the pathogenesis of storiform collagenoma.

The Long and Short of Genome Sequencing: Using a Hybrid Sequencing Strategy to Sequence Oral Microbial Genomes.

Since our chapter on genome sequencing using the GS-FLX pyrosequencer in the First Edition of this book, significant advances have been made in next-generation DNA sequencing (NGS) technology. Not only has the GS-FLX become extinct, but the more recent introduction and establishment of the so-called third-generation DNA sequencers by Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) has revolutionized genomics yet again by generating ultra-long (>100,000 basepair) sequence reads concomitant with an incredible reduction in cost per sequenced basepair. Unfortunately, the ultra-high sequence yields of third-generation sequencers are compromised by their inherent sequencing error rates, prompting an alternative sequencing strategy, i.e., a hybrid sequencing strategy, which combines PacBio/ONT primary datasets with complementary datasets generated by mainstream short-read NGS platforms, e.g., Illumina or Ion Torrent. Although the concept of a hybrid sequencing strategy is not new, existing yields and accuracy of ultra-long and short-read sequencing technologies makes such a strategy achievable, resulting in complete genome sequences in one hit. In this chapter, we describe our updated laboratory and bioinformatic protocols that will allow the average research group to obtain complete oral microbial genome sequences assembled from a combination of DNA sequence data generated by NGS and third-generation platforms.

Bioinformatic Analysis of Natively Paired VH:VL Antibody Repertoires for Antibody Discovery.

Next-generation DNA sequencing (NGS) of human antibody repertoires has been extensively implemented to discover novel antibody drugs, to analyze B-cell developmental features, and to investigate antibody responses to infectious diseases and vaccination. Because the antibody repertoire encoded by human B cells is highly diverse, NGS analyses of antibody genes have provided a new window into understanding antibody responses for basic immunology, biopharmaceutical drug discovery, and immunotherapy. However, many antibody discovery protocols analyze the heavy and light chains separately due to the short-read nature of most NGS technologies, whereas paired heavy and light chain data are required for complete antibody characterization. Here, we describe a computational workflow to process millions of paired antibody heavy and light chain DNA sequence reads using the Illumina MiSeq 2x300 NGS platform. In this workflow, we describe raw NGS read processing and initial quality filtering, the annotation and assembly of antibody clonotypes relating to paired heavy and light chain antibody lineages, and the generation of complete heavy+light consensus sequences for the downstream cloning and expression of human antibody proteins.

Proliferating Pilar Tumors Are Characterized by Recurrent 15q, 6q, and 6p22.2 Alterations.

Proliferating pilar tumors (PPTs) are rare neoplasms of external root sheath derivation, which most commonly occur on the scalp of elderly women. Although typically showing classic histologic features such as trichilemmal type keratinization, a lobular architecture and peripheral palisading, squamous cell carcinoma (SCC) remains a common diagnostic pitfall. Therefore, we sought to explore the molecular pathogenesis of PPTs and compare it with that of cutaneous squamous cell carcinoma (cSCC). Herein, we describe the use of a next-generation DNA sequencing platform to provide the most comprehensive molecular genetic analysis to date of a cohort of 5 PPTs and compare them to 5 head and neck cutaneous SCCs. Recurrent broad arm-level gains of 15q and concurrent single-copy losses of 6q and 6p22.2 were observed in 4 of 5 (80%) PPT cases. Other recurrent mutations or alterations of significance were not found in PPTs. Notably, these chromosomal changes were not identified in any of the 5 cutaneous SCCs, which instead showed recurrent alterations in the known SCC driver genes TP53 , CDKN2A , and NOTCH1 . Here, we show for the first time that PPTs are molecularly distinct from cutaneous SCC and provide evidence that recurrent alterations in chromosome 15 and chromosome 6 are central to the pathogenesis of PPTs.

Recurrent PTPN14 Mutations in Trichilemmoma: Evidence for Distinct Pathways of Molecular Pathogenesis.

Trichilemmoma is a benign cutaneous neoplasm that recapitulates the outer root sheath of the hair follicle. Trichilemmomas may occur sporadically or in association with Cowden syndrome, which is characterized by germline mutations in the lipid phosphatase PTEN (phosphatase and tensin homolog on chromosome 10). Interestingly, most sporadic trichilemmomas do not show PTEN aberrations, but rather activating mutations in HRAS. Despite these important advances, a comprehensive genetic analysis of trichilemmoma has not been reported. Here, we used a next-generation DNA sequencing platform to study 9 sporadic trichilemmoma cases. Seven cases (7/9; 78%) harbored activating mutations in HRAS, consistent with previous findings. Unexpectedly, we identified recurrent mutations in the tyrosine phosphatase PTPN14 (protein tyrosine phosphatase nonreceptor type 14) in 4 cases (4/9; 44%). Three of these cases also harbored HRAS mutations, whereas one case occurred in the absence of a HRAS mutation and showed evidence of biallelic inactivation of PTPN14. Finally, one case (1/9; 11%) showed biallelic inactivation of PTEN in the absence of a HRAS (or PTPN14) mutation. These data suggest at least 3 distinct pathways of molecular pathogenesis in sporadic trichilemmoma and identify PTPN14 as a potentially important contributor to trichilemmoma biology.

Genomic Analyses for Predictors of Response to Chemoradiation in Stage III Non-Small Cell Lung Cancer

BackgroundRadiation with platinum-based chemotherapy is the standard of care for unresectable stage III non-small cell lung cancer (NSCLC). Despite aggressive treatment, progression-free survival and overall survival remain poor. It is unclear whether any tumor genetic mutations are associated with response to chemoradiation therapy. MethodsWe retrospectively reviewed clinical outcomes of patients with stage III NSCLC treated with definitive radiation who had undergone tumor molecular profiling through a next-generation DNA sequencing platform. Cox proportional hazards model was used to investigate associations between clinical outcomes and genetic mutations detected by next-generation sequencing. Results110 patients were identified with stage III NSCLC and underwent definitive radiation between 2013 and 2017 and tumor molecular profiling. Concurrent or sequential chemotherapy was given in 104 patients (95%). Unbiased genomic analyses revealed a significant association between AKT2 mutations and decreased local-regional tumor control and overall survival (hazard ratios [HR] 12.5 and 13.7, P = .003 and P = .003, respectively). Analyses restricted to loss-of-function mutations identified KMT2C and KMT2D deleterious mutations as negative prognostic factors for overall survival (HR 13.4 and 7.0, P < .001 and P < .001, respectively). Deleterious mutations in a panel of 38 DNA damage response and repair pathway genes were associated with improved local-regional control (HR 0.32, P = .049). ConclusionsThis study coupled multiplexed targeted sequencing with clinical outcome and identified mutations in AKT2, KMT2C, and KMT2D as negative predictors of local-regional control and survival, and deleterious mutations in damage response and repair pathway genes were associated with improved local-regional disease control after chemoradiation therapy. These findings will require validation in a larger cohort of patients with prospectively collected and detailed clinical information.

Hedgehog Pathway Alterations Downstream of Patched-1 Are Common in Infundibulocystic Basal Cell Carcinoma

The infundibulocystic variant of basal cell carcinoma (BCC) is characterized histologically by anastamosing strands of basaloid epithelium with associated small infundibular-type cysts. Since its first description in 1987, this rare entity has generated considerable controversy with some authors classifying it as a benign follicular neoplasm rather than a BCC subtype. Prior studies aiming to settle this issue using immunohistochemical analysis reached opposite conclusions. The defining feature of BCC is activation of the Hedgehog signaling pathway, and mutations in Patched-1 (PTCH1) are the most common molecular finding in both sporadic and inherited forms of BCC. Mutations in other downstream components including Smoothened (SMO) and Suppressor of Fused (SUFU) also occur, but are much less common. Here, we report a molecular genetic analysis of a small series of infundibulocystic BCC using a next-generation DNA sequencing platform. All 4 cases harbored mutations or other genetic alterations in components of the Hedgehog pathway, supporting the classification of this entity as a BCC variant. Interestingly, these tumors were enriched for genetic alterations downstream of PTCH1, involving SUFU, SMO, GLI1, and GLI2. This observation was of particular interest given that rare kindreds of the Multiple Hereditary Infundibulocystic BCC syndrome (MHIBCC), which is related, but possibly distinct from the nevoid BCC syndrome, harbored mutations in SUFU. Our results support the classification of the infundibulocystic variant as a subtype of BCC, and suggest that the level at which genetic alterations occur within the Hedgehog pathway may be an important determinant of the morphologic features in BCC.

A novel missense mutation (c.1006C&gt;T) of SPG20 gene associated with Troyer syndrome

The number of gene mutations involved in the hereditary spastic paraplegias is rapidly growing due to the expansion of the frontiers of genomic research by next-generation DNA sequencing platforms. Nevertheless, a comprehensive genetic diagnosis method remains yet unavailable for these diseases. In the current research, an 8-year-old boy with short stature and developmental delay impairment, from a nonconsanguineous family, was referred to our genetic lab. Firstly, based on the physician recommendation, the patient was evaluated by tandem mass spectrometry (MS/MS) for the quantitative examination of amino acids, and then the patient was genetically investigated by karyotype analysis and whole-exome sequencing (WES) technique. Subsequently, targeted Sanger sequencing was applied to confirm the presence of the candidate variant in all the members of the family and screening the other patients for Troyer syndrome. Analysis of inherited metabolic disorders by tandem MS/MS showed the state of all the family members as normal and also karyotyping indicated no chromosomal aberration in the patient. Further investigation by WES technique indicated a homozygous missense variant in the SPG20 gene, c.1006C[T. Targeted sequencing result of the mutation confirmed homozygote state for the affected case and a heterozygote genotype for his parents. The mutation was classified as pathogenic. Detection of novel variants especially pathogenic variantin the SPG20 gene was associated with Troyer syndrome, which encodes a multifunctional protein termed Spartin, assist in improving genotype-phenotype correlation of genetic variants and may facilitate initial diagnosis of Troyer syndrome.

Draft genome sequence of tetracycline-resistant Klebsiella oxytoca CCTCC M207023 producing 2,3-butanediol isolated from China.

Recently, the Gram-negative bacterium Klebsiella oxytoca has been identified as an emerging pathogen. Here we report the draft genome of a 2,3-butanediol-producing strain, K. oxytoca CCTCC M207023, isolated from soil in Nanjing, China. The tetracycline-resistant phenotype and the high yield of 2,3-butanediol was demonstrated. The draft genome of K. oxytoca CCTCC M207023 was determined using an Illumina NovaSeq™ 6000 next-generation DNA sequencing platform. Clean sequencing data were subsequently assembled using SOAPdenovo. The draft genome of K. oxytoca CCTCC M207023, comprising 5 658 144bp and with a GC content of 56.50%, was assembled into 5262 open-reading frames (ORFs). Antimicrobial resistance genes were also annotated. The draft genome sequence of K. oxytoca CCTCC M207023 reported here will be a reference for comparative analysis with the antimicrobial resistance mechanisms for the safety of 2,3-butanediol industrial production.

Morphologic, Immunohistochemical, and Molecular Distinction Between Fibroepithelioma of Pinkus and "Fenestrated" Basal Cell Carcinoma.

Fibroepithelioma of Pinkus (FEP) is a rare cutaneous neoplasm with a characteristic fenestrated architecture and a prominent spindle cell stromal component and which invariably pursues an indolent course. The classification of FEP has been much debated since its first description in 1953, with some arguing that it represents a variant of a basal cell carcinoma (BCC) while others view it as a variant of a trichoblastoma. Multiple previous immunohistochemical studies aiming to clarify this issue have yielded conflicting results. To date, there have been no molecular studies of FEP. We identified 16 cases of fenestrated follicular neoplasms and classified them as BCC or FEP based solely on histomorphologic criteria. CK20 immunohistochemistry supported this classification scheme, with FEP showing significantly more CK20-positive Merkel cells than BCC. We then analyzed a subset of these tumors by a targeted next-generation DNA sequencing platform. All the BCC cases harbored pathogenic PTCH1 mutations, confirming the diagnosis. By contrast, none of the FEP cases harbored a PTCH1 mutation or indeed any mutation known to be causally linked to the development of BCC. Our results suggest that FEP can be distinguished from BCC on morphologic, immunohistochemical, and molecular genetic grounds. We argue that FEP is better considered a benign follicular neoplasm and support its classification as a variant of trichoblastoma.

Challenges of Single-Molecule DNA Sequencing with Solid-State Nanopores.

A powerful DNA sequencing tool with high accuracy, long read length and high-throughput would be required more and more for decoding the complicated genetic code. Solid-state nanopore has attracted many researchers for its promising future as a next-generation DNA sequencing platform due to the processability, the robustness and the large-scale integratability. While the diverse materials have been widely explored for a solid-state nanopore, silicon nitride (Si3N4) is especially preferable from the viewpoint of mass production based on semiconductor fabrication process. Here, as a nanopore sensing mechanism, we focused on the ionic blockade current method which is the most developed technique. We also highlight the main challenges of Si3N4 nanopore-based DNA sequencer that should be addressed: the fabrication of ultra-small nanopore and ultra-thin membrane, the modulation of DNA translocation speed and the detection of base-specific signals. In this chapter, we discuss the recent progress relating to solid-state nanopore DNA sequencing, which helps to provide a comprehensive information about the current technical situation.

Draft genome sequence of Bacillus sp. M13(2017), a multidrug-resistant subclass B1 blaNDM-producing, spore-forming bacterium isolated from China.

Bacillus spp., which have relevance to pseudo-epidemics in clinics, may cause serious infections as well as high morbidity and mortality in hospitalised patients with haematological malignancies. Here we report the isolation of Bacillus sp. M13(2017) with a multidrug-resistant phenotype and spore-producing structures, a new strain isolated from radiation-polluted soil in Xinjiang Uyghur Autonomous Region, China. The draft genome of Bacillus sp. M13(2017) was obtained using an Illumina HiSeqTM 2000 next-generation DNA sequencing platform. Subsequently, clean sequencing data were assembled using SOAPdenovo and were optimised using GapCloser. The draft genome sequence of Bacillus sp. M13(2017) includes 5873661bp, a GC content of 34.84% and is assembled into 6108 open reading frames, of which ca. 5.08% and 1.76% are related to spore formation and antimicrobial resistance, respectively. The blaNDM gene encoding a subclass B1 metallo-β-lactamase was also discovered in this isolate. The genome sequence of Bacillus sp. M13(2017) reported here will be a reference for comparative analysis with genomic and clinically important multidrug resistance features of Bacillus sp.

Sequencing, Assembling, and Finishing Complete Bacteriophage Genomes.

Next-generation DNA sequencing (NGS) technologies have made generating genomic sequence for organisms of interest affordable and commonplace. However, NGS platforms and analysis software are generally tuned to be used on large and complex genomes or metagenomic samples. Determining the complete genome sequence of a single bacteriophage requires a somewhat different perspective, workflow, and sensitivity to the nature of phages. Because phage genomes consist of mostly coding regions (see Pope/Jacobs-Sera chapter), a very high standard should be adopted when completing these genomes so that the subsequent steps of annotation and analysis are not sabotaged by sequencing errors. While read quality and assembly algorithms have continued to improve, achieving this standard still requires a significant amount of human oversight and expertise. This chapter describes our workflow for sequencing, assembling, and finishing phage genomes to a high standard by the NGS platforms Illumina, Ion Torrent, and 454.

Chromatin Immunoprecipitation (ChIP) in Schizosaccharomyces pombe.

Chromatin immunoprecipitation (ChIP), the cross-linking of chromatin followed by immunoprecipitation with antibodies against a chromatin target, is a key method for measuring association of proteins with a specific genomic region(s). As a negative control, a mock ChIP experiment in which no antibody is added to the immunoprecipitation reaction is included. Enriched DNA fragments from a ChIP experiment can be analyzed in a variety of ways. For semiquantitative analysis, a region of interest can be amplified using standard polymerase chain reaction (PCR) techniques. PCR products are analyzed on agarose (or polyacrylamide) gels and band intensity calculated with a standard imaging software. ChIP enrichment is usually calculated as the ratio of ChIP to input compared with a similar ratio for a reference region not expected to be enriched for that factor. For heterochromatin analysis, housekeeping genes such as act1+ are good references. Real-time quantitative PCR (qPCR) can be used for a fully quantitative approach. If a factor is to be mapped across the genome, ChIP DNA can be amplified and labeled for microarray analysis or scrutinized on a next-generation DNA sequencing platform.

Peptide Synthesis on a Next-Generation DNA Sequencing Platform.

Methods for displaying large numbers of peptides on solid surfaces are essential for high-throughput characterization of peptide function and binding properties. Here we describe a method for converting the >10(7) flow cell-bound clusters of identical DNA strands generated by the Illumina DNA sequencing technology into clusters of complementary RNA, and subsequently peptide clusters. We modified the flow-cell-bound primers with ribonucleotides thus enabling them to be used by poliovirus polymerase 3D(pol) . The primers hybridize to the clustered DNA thus leading to RNA clusters. The RNAs fold into functional protein- or small molecule-binding aptamers. We used the mRNA-display approach to synthesize flow-cell-tethered peptides from these RNA clusters. The peptides showed selective binding to cognate antibodies. The methods described here provide an approach for using DNA clusters to template peptide synthesis on an Illumina flow cell, thus providing new opportunities for massively parallel peptide-based assays.

Splicing Express: a software suite for alternative splicing analysis using next-generation sequencing data.

Motivation. Alternative splicing events (ASEs) are prevalent in the transcriptome of eukaryotic species and are known to influence many biological phenomena. The identification and quantification of these events are crucial for a better understanding of biological processes. Next-generation DNA sequencing technologies have allowed deep characterization of transcriptomes and made it possible to address these issues. ASEs analysis, however, represents a challenging task especially when many different samples need to be compared. Some popular tools for the analysis of ASEs are known to report thousands of events without annotations and/or graphical representations. A new tool for the identification and visualization of ASEs is here described, which can be used by biologists without a solid bioinformatics background.Results. A software suite named Splicing Express was created to perform ASEs analysis from transcriptome sequencing data derived from next-generation DNA sequencing platforms. Its major goal is to serve the needs of biomedical researchers who do not have bioinformatics skills. Splicing Express performs automatic annotation of transcriptome data (GTF files) using gene coordinates available from the UCSC genome browser and allows the analysis of data from all available species. The identification of ASEs is done by a known algorithm previously implemented in another tool named Splooce. As a final result, Splicing Express creates a set of HTML files composed of graphics and tables designed to describe the expression profile of ASEs among all analyzed samples. By using RNA-Seq data from the Illumina Human Body Map and the Rat Body Map, we show that Splicing Express is able to perform all tasks in a straightforward way, identifying well-known specific events.Availability and Implementation.Splicing Express is written in Perl and is suitable to run only in UNIX-like systems. More details can be found at: http://www.bioinformatics-brazil.org/splicingexpress.

Microbial metagenomics of oil sands tailings ponds: small bugs, big data.

Modern molecular biology techniques, particularly new DNA sequencing methods, have opened our eyes to the fact that microorganisms live in an extraordinary range of natural environments, from desert soils and boiling hot springs to Antarctic ice, in ancient sea beds and under the surface of some rocks, inside leaves and in the human gut, among others. They also colonize manmade environments, such as the enormous oil sands tailings ponds in northeastern Alberta, Canada, that are commonly reported by the press as being toxic to all life. Although the tailings ponds may not be hospitable to higher organisms, all eight ponds that we have analyzed to date (comprising nearly 100 samples) harbour large, diverse, and complex communities of microbes (An et al. 2013; Penner and Foght 2010). In fact, we have found that microbes typically are present at about 1 million cells per millilitre of oil sands tailings, which scales to a total of about a sextillion (10 21 ) cells in the nearly 1 billion cubic metres of tailings currently retained in the Athabasca Oil Sands region. That approaches the estimated number of stars in the Universe! This begs the questions: Which microbes live in the tailings ponds? What are they doing there? Where did they come from? Are they important in situ, either impacting current tailings management or eventual reclamation strategies for the tailings ponds? To address such questions required substantial long-term funding to develop methods for analyzing large suites of refractory samples using nextgeneration DNA sequencing platforms. Genome Canada and corporate partners from the energy industry provided that support via “Metagenomics for Greener Production and Extraction of Hydrocarbon Energy” (the Hydrocarbon Metagenomics Project; www.hydrocarbonmetagenomics.com). The project investigated several aspects of fossil fuel production, including coal bed methane production and souring of conventional petroleum reservoirs, but this presentation focuses on oil sands. Media images of tailings ponds typically are aerial photographs showing murky water and floating bitumen. However, beneath that shallow water layer lies the vast bulk of the tailings: a thick gel-like suspension of mineral and organic waste generated by aqueous extraction of surface-mined oil sands ores. The ores primarily comprise sand, fine clay particles, and adherent bitumen (a viscous crude oil) that is extracted by using hot alkaline water plus hydrocarbon solvents such as naphtha, a refined product similar to kerosene and comprising hundreds of individual compounds. Most of the solvent (>99%) is recovered after extraction and re-used. The waste from the extraction process comprises a thin slurry of water, sand, clay, unextracted bitumen, and the unrecovered solvent: this is the material deposited and retained in the tailings ponds pending reclamation. The sand settles out of suspension rapidly and is collected for other purposes, but the suspended clays settle extremely slowly, requiring years or decades to achieve the consistency of toothpaste while the water (and some bitumen) rises to the pond surface for re-use. Meanwhile, microbes indigenous to the ores and the water that have survived the extraction process and those that are introduced through rainfall and blowing dust become established in the ponds. Because the vast majority of material in the tailings ponds is anaerobic, most of the microbes colonizing the ponds grow without access to oxygen (via sulfate reduction or methane generation) and some are even killed by exposure to air. What are these microbes doing in the ponds? The community composition and activity are dictated by the carbon sources available to support microbial growth. The predominant mass of organic material in the ponds is unextracted bitumen, but, because it is virtually nondegradable (like asphalt), it is not a significant carbon source for the microbes. Instead, the much smaller proportion of unrecovered solvent entrained in the fresh tailings is the major carbon input supporting growth of the indigenous microbes. More specifically, only certain components of the solvents are degraded under anaerobic conditions, typically only slowly over months or years.

Genetic-geographic correlation revealed across a broad European ecotypic sample of perennial ryegrass (Lolium perenne) using array-based SNP genotyping.

Key messagePublically available SNP array increases the marker density for genotyping of forage crop,Lolium perenne. Applied to 90 European ecotypes composed of 716 individuals identifies a significant genetic–geographic correlation. Grassland ecosystems are ubiquitous across temperate and tropical regions, totalling 37 % of the terrestrial land cover of the planet, and thus represent a global resource for understanding local adaptations to environment. However, genomic resources for grass species (outside cereals) are relatively poor. The advent of next-generation DNA sequencing and high-density SNP genotyping platforms enables the development of dense marker assays for population genetics analyses and genome-wide association studies. A high-density SNP marker resource (Illumina Infinium assay) for perennial ryegrass (Lolium perenne) was created and validated in a broad ecotype collection of 716 individuals sampled from 90 sites across Europe. Genetic diversity within and between populations was assessed. A strong correlation of geographic origin to genetic structure was found using principal component analysis, with significant correlation to longitude and latitude (P < 0.001). The potential of this array as a resource for studies of germplasm diversity and identifying traits underpinning adaptive variation is highlighted.Electronic supplementary materialThe online version of this article (doi:10.1007/s00122-015-2556-3) contains supplementary material, which is available to authorized users.

Summary of the advanced virus detection technologies users group efforts-2013.

An informal consortium of scientists from the biopharmaceutical industry, academia, and government agencies, including regulators, coalesced during 2013 to further explore advanced virus detection technologies or appropriate applications for characterization and evaluation of biologicals. As a Parenteral Drug Association task force, the Users Group came to focus on four key work areas that required better understanding and data generation: (1) evaluation of sample preparation and processing steps for different sample types, (2) determination of method sensitivity by performing spike recovery studies using selected virus stocks, (3) development of a reliable and comprehensive viral sequence database, and (4) evaluation of bioinformatic analysis pipelines, with primary focus on next-generation DNA sequencing platforms. The year of monthly working meetings and additional subgroup discussions culminated in the 2013 PDA/FDA Advanced Technologies for Virus Detection in the Evaluation of Biologicals Conference held November 13-14, 2013 in Bethesda, MD, which provided a forum for group participants and others for data sharing and knowledge exchange. The Users Group has continued its efforts during 2014 as a PDA Interest Group with increased participation from technology developers and contract research organizations.

EST sequencing and gene expression profiling in Scutellaria baicalensis.

Scutellaria baicalensis is an important medicinal plant, but few genomic resources are available for this species, as well as for other non-model plants. One of the major new directions in genome research is to discover the full spectrum of genes transcribed from the whole genome. Here, we report extensive transcriptome data of the early growth stage of S. baicalensis. This transcriptome consensus sequence was constructed by de novo assembly of shotgun sequencing data, obtained using multiple next-generation DNA sequencing (NGS) platforms (Roche/454 GS_FLX+ and Illumina/Solexa HiSeq2000). We show that this new approach to obtain extensive mRNA is an efficient strategy for genome-wide transcriptome analysis. We obtained 1,226,938 and 161,417,646 reads using the GS_FLX and the Illumina/Solexa HiSeq2000, respectively. De novo assembly of the high-quality GS_FLX and Illumina reads (95 % and 75 %) resulted in more than 82 Mb of mRNA consensus sequence, which we assembled into 51,188 contigs, with at least 500 bp per contig. Of these contigs, 39,581 contained known genes, as determined by BLASTX searches against non-redundant NCBI database. Of these, 20,498 different genes were expressed during the early growth stage of S. baicalensis. We have made the expressed sequences available on a public database. Our results demonstrate the utility of combining NGS technologies as a basis for the development of genomic tools in non-model, medicinal plant species. Knowledge of all described genes and quantitation of the expressed genes, including the transcription factors involved, will be useful in studies of the biology of S. baicalensis gene regulation.