Peregrine: A rapid and unbiased method to produce strand-specific RNA-Seq libraries from small quantities of starting material.

Use of second generation sequencing (SGS) technologies for transcriptional profiling (RNA-Seq) has revolutionized transcriptomics, enabling measurement of RNA abundances with unprecedented specificity and sensitivity and the discovery of novel RNA species. Preparation of RNA-Seq libraries requires conversion of the RNA starting material into cDNA flanked by platform-specific adaptor sequences. Each of the published methods and commercial kits currently available for RNA-Seq library preparation suffers from at least one major drawback, including long processing times, large starting material requirements, uneven coverage, loss of strand information and high cost. We report the development of a new RNA-Seq library preparation technique that produces representative, strand-specific RNA-Seq libraries from small amounts of starting material in a fast, simple and cost-effective manner. Additionally, we have developed a new quantitative PCR-based assay for precisely determining the number of PCR cycles to perform for optimal enrichment of the final library, a key step in all SGS library preparation workflows.

Systematic Bioinformatics Analysis Based on Public and Second-Generation Sequencing Transcriptome Data: A Study on the Diagnostic Value and Potential Mechanisms of Immune-Related Genes in Acute Myocardial Infarction.

Acute myocardial infarction (AMI) is one of the most serious cardiovascular diseases worldwide. Advances in genomics have provided new ideas for the development of novel molecular biomarkers of potential clinical value for AMI. Methods: Based on microarray data from a public database, differential analysis and functional enrichment analysis were performed to identify aberrantly expressed genes in AMI and their potential functions. CIBERSORT was used for immune landscape analysis. We also obtained whole blood samples of 3 patients with AMI and performed second-generation sequencing (SGS) analysis. Weighted gene co-expression network analysis (WGCNA) and cross-tabulation analysis identified AMI-related key genes. Receiver operating characteristic (ROC) curves were used to assess the diagnostic power of key genes. Single-gene gene set enrichment analysis (GSEA) revealed the molecular mechanisms of diagnostic indicators. Results: A total of 53 AMI-related DEGs from a public database were obtained and found to be involved in immune cell activation, immune response regulation, and cardiac developmental processes. CIBERSORT confirmed that the immune microenvironment was altered between AMI and normal samples. A total of 77 hub genes were identified by WGCNA, and 754 DEGs were obtained from own SGS data. Seven diagnostic indicators of AMI were obtained, namely GZMA, NKG7, TBX21, TGFBR3, SMAD7, KLRC4, and KLRD1. The single-gene GSEA suggested that the diagnostic indicators seemed to be closely implicated in cell cycle, immune response, cardiac developmental, and functional regulatory processes. Conclusion: The present study provides new diagnostic indicators for AMI and further confirms the feasibility of the results of genome-wide gene expression analysis.

Analyzing Modern Biomolecules: The Revolution of Nucleic-Acid Sequencing - Review.

Recent developments have revolutionized the study of biomolecules. Among them are molecular markers, amplification and sequencing of nucleic acids. The latter is classified into three generations. The first allows to sequence small DNA fragments. The second one increases throughput, reducing turnaround and pricing, and is therefore more convenient to sequence full genomes and transcriptomes. The third generation is currently pushing technology to its limits, being able to sequence single molecules, without previous amplification, which was previously impossible. Besides, this represents a new revolution, allowing researchers to directly sequence RNA without previous retrotranscription. These technologies are having a significant impact on different areas, such as medicine, agronomy, ecology and biotechnology. Additionally, the study of biomolecules is revealing interesting evolutionary information. That includes deciphering what makes us human, including phenomena like non-coding RNA expansion. All this is redefining the concept of gene and transcript. Basic analyses and applications are now facilitated with new genome editing tools, such as CRISPR. All these developments, in general, and nucleic-acid sequencing, in particular, are opening a new exciting era of biomolecule analyses and applications, including personalized medicine, and diagnosis and prevention of diseases for humans and other animals.

Assessment of Genetic Diversity and Structure of Large Garlic (Allium sativum) Germplasm Bank, by Diversity Arrays Technology "Genotyping-by-Sequencing" Platform (DArTseq).

Garlic (Allium sativum) is used worldwide in cooking and industry, including pharmacology/medicine and cosmetics, for its interesting properties. Identifying redundancies in germplasm blanks to generate core collections is a major concern, mostly in large stocks, in order to reduce space and maintenance costs. Yet, similar appearance and phenotypic plasticity of garlic varieties hinder their morphological classification. Molecular studies are challenging, due to the large and expected complex genome of this species, with asexual reproduction. Classical molecular markers, like isozymes, RAPD, SSR, or AFLP, are not convenient to generate germplasm core-collections for this species. The recent emergence of high-throughput genotyping-by-sequencing (GBS) approaches, like DArTseq, allow to overcome such limitations to characterize and protect genetic diversity. Therefore, such technology was used in this work to: (i) assess genetic diversity and structure of a large garlic-germplasm bank (417 accessions); (ii) create a core collection; (iii) relate genotype to agronomical features; and (iv) describe a cost-effective method to manage genetic diversity in garlic-germplasm banks. Hierarchical-cluster analysis, principal-coordinates analysis and STRUCTURE showed general consistency, generating three main garlic-groups, mostly determined by variety and geographical origin. In addition, high-resolution genotyping identified 286 unique and 131 redundant accessions, used to select a reduced size germplasm-bank core collection. This demonstrates that DArTseq is a cost-effective method to analyze species with large and expected complex genomes, like garlic. To the best of our knowledge, this is the first report of high-throughput genotyping of a large garlic germplasm. This is particularly interesting for garlic adaptation and improvement, to fight biotic and abiotic stresses, in the current context of climate change and global warming.

Second-generation sequencing of forensic STRs using the Ion Torrent™ HID STR 10-plex and the Ion PGM™.

Second-generation sequencing (SGS) using Roche/454 and Illumina platforms has proved capable of sequencing the majority of the key forensic genetic STR systems. Given that Roche has announced that the 454 platforms will no longer be supported from 2015, focus should now be shifted to competing SGS platforms, such as the MiSeq (Illumina) and the Ion Personal Genome Machine (Ion PGM™; Thermo Fisher). There are currently several challenges faced with amplicon-based SGS STR typing in forensic genetics, including current lengths of amplicons for CE-typing and lack of uniform data analysis between laboratories. Thermo Fisher has designed a human identification (HID) short tandem repeat (STR) 10-plex panel including amelogenin, CSF1PO, D16S539, D3S1358, D5S818, D7S820, D8S1179, TH01, TPOX and vWA, where the primers have been designed specifically for the purpose of SGS and the data analysis is supported by Ion Torrent™ software. Hence, the combination of the STR 10-plex and the Ion PGM™ represents the first fully integrated SGS STR typing solution from PCR to data analysis. In this study, four experiments were performed to evaluate the alpha-version of the STR 10-plex: (1) typing of control samples; (2) analysis of sensitivity; (3) typing of mixtures; and (4) typing of biological crime case samples. Full profiles and concordant results between replicate SGS runs and CE-typing were observed for all control samples. Full profiles were seen with DNA input down to 50 pg, with the exception of a single locus drop-out in one of the 100 pg dilutions. Mixtures were easily deconvoluted down to 20:1, although alleles from the minor contributor had to be identified manually as some signals were not called by the Ion Torrent™ software. Interestingly, full profiles were obtained for all biological samples from real crime and identification cases, in which only partial profiles were obtained with PCR-CE assays. In conclusion, the Ion Torrent™ HID STR 10-plex panel offers an all-in-one solution from amplification of STRs and amelogenin, and sequencing to data analysis.

Development and validation of an rDNA operon based primer walking strategy applicable to de novo bacterial genome finishing.

Advances in sequencing technology have drastically increased the depth and feasibility of bacterial genome sequencing. However, little information is available that details the specific techniques and procedures employed during genome sequencing despite the large numbers of published genomes. Shotgun approaches employed by second-generation sequencing platforms has necessitated the development of robust bioinformatics tools for in silico assembly, and complete assembly is limited by the presence of repetitive DNA sequences and multi-copy operons. Typically, re-sequencing with multiple platforms and laborious, targeted Sanger sequencing are employed to finish a draft bacterial genome. Here we describe a novel strategy based on the identification and targeted sequencing of repetitive rDNA operons to expedite bacterial genome assembly and finishing. Our strategy was validated by finishing the genome of Paenibacillus polymyxa strain CR1, a bacterium with potential in sustainable agriculture and bio-based processes. An analysis of the 38 contigs contained in the P. polymyxa strain CR1 draft genome revealed 12 repetitive rDNA operons with varied intragenic and flanking regions of variable length, unanimously located at contig boundaries and within contig gaps. These highly similar but not identical rDNA operons were experimentally verified and sequenced simultaneously with multiple, specially designed primer sets. This approach also identified and corrected significant sequence rearrangement generated during the initial in silico assembly of sequencing reads. Our approach reduces the required effort associated with blind primer walking for contig assembly, increasing both the speed and feasibility of genome finishing. Our study further reinforces the notion that repetitive DNA elements are major limiting factors for genome finishing. Moreover, we provided a step-by-step workflow for genome finishing, which may guide future bacterial genome finishing projects.