High-throughput Sequencing Sample Research Articles

A Step Towards Validation of High-Throughput Sequencing for the Identification of Plant Pathogenic Oomycetes.

The advancement in high-throughput sequencing (HTS) technology allows the detection of pathogens without the need for isolation or template amplification. Plant regulatory agencies worldwide are adopting HTS as a prescreening tool for plant pathogens in imported plant germplasm. The technique is a multipronged process and, often, the bioinformatic analysis complicates detection. Previously, we developed E-probe diagnostic nucleic acid analysis (EDNA), a bioinformatic tool that detects pathogens in HTS data. EDNA uses custom databases of signature nucleic acid sequences (e-probes) to reduce computational effort and subjectivity when determining pathogen presence in a sample. E-probes of Pythium ultimum and Phytophthora ramorum were previously validated only using simulated HTS data. However, HTS samples generated from infected hosts or pure culture may vary in pathogen concentration, sequencing bias, and data quality, suggesting that each pathosystem requires further validation. Here, we used metagenomic and genomic HTS data generated from infected hosts and pure culture, respectively, to further validate and curate e-probes of Pythium ultimum and Phytophthora ramorum. E-probe length was found to be a determinant of diagnostic sensitivity and specificity; 80-nucleotide e-probes increased the diagnostic specificity to 100%. Curating e-probes to increase specificity affected diagnostic sensitivity only for 80-nucleotide Pythium ultimum e-probes. Comparing e-probes with alternative databases and bioinformatic tools in their speed and ability to find Pythium ultimum and Phytophthora ramorum demonstrated that, although pathogen sequence reads were detected by other methods, they were less specific and slower when compared with e-probes.

Breaking Barriers with Bread: Using the Sourdough Starter Microbiome to Teach High-Throughput Sequencing Techniques.

ABSTRACTWidespread usage of high-throughput sequencing (HTS) in the LIFE SCIENCES has produced a demand for undergraduate and graduate institutions to offer classes exposing students to all aspects of HTS (sample acquisition, laboratory work, sequencing technologies, bioinformatics, and statistical analyses). Despite the increase in demand, many challenges exist for these types of classes. We advocate for the usage of the sourdough starter microbiome for implementing meta-amplicon sequencing. The relatively small community, dominated by a few taxa, enables potential contaminants to be easily identified, while between-sample differences can be quickly statistically assessed. Finally, bioinformatic pipelines and statistical analyses can be carried out on personal student laptops or in a teaching computer lab. In two semesters adopting this system, 12 of 14 students were able to effectively capture the sourdough starter microbiome, using the instructor’s paired sample as reference.

Contaminating viral sequences in high-throughput sequencing viromics: a linkage study of 700 sequencing libraries

ObjectivesSample preparation for high-throughput sequencing (HTS) includes treatment with various laboratory components, potentially carrying viral nucleic acids, the extent of which has not been thoroughly investigated. Our aim was to systematically examine a diverse repertoire of laboratory components used to prepare samples for HTS in order to identify contaminating viral sequences. MethodsA total of 322 samples of mainly human origin were analysed using eight protocols, applying a wide variety of laboratory components. Several samples (60% of human specimens) were processed using different protocols. In total, 712 sequencing libraries were investigated for viral sequence contamination. ResultsAmong sequences showing similarity to viruses, 493 were significantly associated with the use of laboratory components. Each of these viral sequences had sporadic appearance, only being identified in a subset of the samples treated with the linked laboratory component, and some were not identified in the non-template control samples. Remarkably, more than 65% of all viral sequences identified were within viral clusters linked to the use of laboratory components. ConclusionsWe show that high prevalence of contaminating viral sequences can be expected in HTS-based virome data and provide an extensive list of novel contaminating viral sequences that can be used for evaluation of viral findings in future virome and metagenome studies. Moreover, we show that detection can be problematic due to stochastic appearance and limited non-template controls. Although the exact origin of these viral sequences requires further research, our results support laboratory-component-linked viral sequence contamination of both biological and synthetic origin.

Effect of dietary olive oil and palm oil on rumen bacterial composition in dairy cows

The rumen bacteria play a major role in lipid metabolism. Bacteria remove the double bonds of unsaturated fatty acids resulting in the production of saturated fatty acids, which are incorporated in milk. Crude olive oil (rich in unsaturated fatty acids) represents a potentially valuable feed source for dairy cows that might enhance the human-health beneficial composition of milk and dairy products. This project studied the effect of supplementing dairy cow diets with olive oil (OO) and palm oil (HVO) on rumen microbiota. For 63 days the animals were fed a control diet (basal diet) with no added lipid and two fat-supplemented diets (30 g kg−1 DM). Rumen sampling were performed at the onset of the experiment and every 21 days for 63 days using a rumen scoop. Total microbial DNA was extracted from ruminal samples for high-throughput sequencing of the 16S rRNA gene through Illumina MiSeq platform. Results revealed the dominance of phyla Firmicutes and Bacteroidetes. Firmicutes was the most prevalent phyla in diet control (75.2 %), OO (71.1 %) and HVO (75.2 %). At genus level Succiniclasticum and Prevotella were the dominant genera, belonging to Firmicutes and Bacteroidetes respectively. Succiniclasticum decrease significantly their relative abundance during OO supplementation (p)

Culture independent analysis using gnd as a target gene to assess Escherichia coli diversity and community structure

Current culture methods to investigate changes in Escherichia coli community structure are often slow and laborious. Genes such as gnd (6-phosphogluconate dehydrogenase) have a highly variable nucleotide sequence and may provide a target for E. coli microbiome analysis using culture-independent methods. Metabarcoded PCR primers were used to generate separate libraries from calf faecal samples for high throughput sequencing. Although a total of 348 separate gnd sequence types (gSTs) were identified, 188 were likely to be due to sequencing errors. Of the remaining 160 gSTs, 92 did not match those in a database of 319 separate gnd sequences. ‘Animal’ was the main determinant of E. coli diversity with limited impact of sample type or DNA extraction method on intra-host E. coli community variation from faeces and recto-anal mucosal swab samples. This culture-independent study has addressed the difficulties of quantifying bacterial intra-species diversity and revealed that, whilst individual animals may harbour >50 separate E. coli strains, communities are dominated by <10 strains alongside a large pool of subdominant strains present at low abundances. This method will be useful for characterising the diversity and population structure of E. coli in experimental studies designed to assess the impact of interventions on the gut microbiome.

Microdissection of lampbrush chromosomes as an approach for generation of locus-specific FISH-probes and samples for high-throughput sequencing.

BackgroundOver the past two decades, chromosome microdissection has been widely used in diagnostics and research enabling analysis of chromosomes and their regions through probe generation and establishing of chromosome- and chromosome region-specific DNA libraries. However, relatively small physical size of mitotic chromosomes limited the use of the conventional chromosome microdissection for investigation of tiny chromosomal regions.ResultsIn the present study, we developed a workflow for mechanical microdissection of giant transcriptionally active lampbrush chromosomes followed by the preparation of whole-chromosome and locus-specific fluorescent in situ hybridization (FISH)-probes and high-throughput sequencing. In particular, chicken (Gallus g. domesticus) lampbrush chromosome regions as small as single chromomeres, individual lateral loops and marker structures were successfully microdissected. The dissected fragments were mapped with high resolution to target regions of the corresponding lampbrush chromosomes. For investigation of RNA-content of lampbrush chromosome structures, samples retrieved by microdissection were subjected to reverse transcription. Using high-throughput sequencing, the isolated regions were successfully assigned to chicken genome coordinates. As a result, we defined precisely the loci for marker structures formation on chicken lampbrush chromosomes 2 and 3. Additionally, our data suggest that large DAPI-positive chromomeres of chicken lampbrush chromosome arms are characterized by low gene density and high repeat content.ConclusionsThe developed technical approach allows to obtain DNA and RNA samples from particular lampbrush chromosome loci, to define precisely the genomic position, extent and sequence content of the dissected regions. The data obtained demonstrate that lampbrush chromosome microdissection provides a unique opportunity to correlate a particular transcriptional domain or a cytological structure with a known DNA sequence. This approach offers great prospects for detailed exploration of functionally significant chromosomal regions.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2437-4) contains supplementary material, which is available to authorized users.