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Concept: Sequence

331

We report on the sequencing of 10,545 human genomes at 30×-40× coverage with an emphasis on quality metrics and novel variant and sequence discovery. We find that 84% of an individual human genome can be sequenced confidently. This high-confidence region includes 91.5% of exon sequence and 95.2% of known pathogenic variant positions. We present the distribution of over 150 million single-nucleotide variants in the coding and noncoding genome. Each newly sequenced genome contributes an average of 8,579 novel variants. In addition, each genome carries on average 0.7 Mb of sequence that is not found in the main build of the hg38 reference genome. The density of this catalog of variation allowed us to construct high-resolution profiles that define genomic sites that are highly intolerant of genetic variation. These results indicate that the data generated by deep genome sequencing is of the quality necessary for clinical use.

Concepts: Sequence, Genome, Human Genome Project, Human genome, Genetics, Gene, Genomics, DNA

234

To investigate cognitive operations underlying sequential problem solving, we confronted ten Goffin’s cockatoos with a baited box locked by five different inter-locking devices. Subjects were either naïve or had watched a conspecific demonstration, and either faced all devices at once or incrementally. One naïve subject solved the problem without demonstration and with all locks present within the first five sessions (each consisting of one trial of up to 20 minutes), while five others did so after social demonstrations or incremental experience. Performance was aided by species-specific traits including neophilia, a haptic modality and persistence. Most birds showed a ratchet-like progress, rarely failing to solve a stage once they had done it once. In most transfer tests subjects reacted flexibly and sensitively to alterations of the locks' sequencing and functionality, as expected from the presence of predictive inferences about mechanical interactions between the locks.

Concepts: Future, Psychology, Problem, Sequence, Educational psychology, Demonstration, How to Solve It, Problem solving

222

Motor skill memory is first encoded online in a fragile form during practice and then converted into a stable form by offline consolidation, which is the behavioral stage critical for successful learning. Praise, a social reward, is thought to boost motor skill learning by increasing motivation, which leads to increased practice. However, the effect of praise on consolidation is unknown. Here, we tested the hypothesis that praise following motor training directly facilitates skill consolidation. Forty-eight healthy participants were trained on a sequential finger-tapping task. Immediately after training, participants were divided into three groups according to whether they received praise for their own training performance, praise for another participant’s performance, or no praise. Participants who received praise for their own performance showed a significantly higher rate of offline improvement relative to other participants when performing a surprise recall test of the learned sequence. On the other hand, the average performance of the novel sequence and randomly-ordered tapping did not differ between the three experimental groups. These results are the first to indicate that praise-related improvements in motor skill memory are not due to a feedback-incentive mechanism, but instead involve direct effects on the offline consolidation process.

Concepts: Practice, Real analysis, Series, Sequence, Learning, Psychology, Training, Skill

185

Next-generation sequencing (NGS) is increasingly being adopted as the backbone of biomedical research. With the commercialization of various affordable desktop sequencers, NGS will be reached by increasing numbers of cellular and molecular biologists, necessitating community consensus on bioinformatics protocols to tackle the exponential increase in quantity of sequence data. The current resources for NGS informatics are extremely fragmented. Finding a centralized synthesis is difficult. A multitude of tools exist for NGS data analysis; however, none of these satisfies all possible uses and needs. This gap in functionality could be filled by integrating different methods in customized pipelines, an approach helped by the open-source nature of many NGS programmes. Drawing from community spirit and with the use of the Wikipedia framework, we have initiated a collaborative NGS resource: The NGS WikiBook. We have collected a sufficient amount of text to incentivize a broader community to contribute to it. Users can search, browse, edit and create new content, so as to facilitate self-learning and feedback to the community. The overall structure and style for this dynamic material is designed for the bench biologists and non-bioinformaticians. The flexibility of online material allows the readers to ignore details in a first read, yet have immediate access to the information they need. Each chapter comes with practical exercises so readers may familiarize themselves with each step. The NGS WikiBook aims to create a collective laboratory book and protocol that explains the key concepts and describes best practices in this fast-evolving field.

Concepts: Sequence, Sustainability, Biology, Medical research, Bench, Exponential growth, Molecular biology, Wikipedia

181

Background For more than a decade, risk stratification for hypertrophic cardiomyopathy has been enhanced by targeted genetic testing. Using sequencing results, clinicians routinely assess the risk of hypertrophic cardiomyopathy in a patient’s relatives and diagnose the condition in patients who have ambiguous clinical presentations. However, the benefits of genetic testing come with the risk that variants may be misclassified. Methods Using publicly accessible exome data, we identified variants that have previously been considered causal in hypertrophic cardiomyopathy and that are overrepresented in the general population. We studied these variants in diverse populations and reevaluated their initial ascertainments in the medical literature. We reviewed patient records at a leading genetic-testing laboratory for occurrences of these variants during the near-decade-long history of the laboratory. Results Multiple patients, all of whom were of African or unspecified ancestry, received positive reports, with variants misclassified as pathogenic on the basis of the understanding at the time of testing. Subsequently, all reported variants were recategorized as benign. The mutations that were most common in the general population were significantly more common among black Americans than among white Americans (P<0.001). Simulations showed that the inclusion of even small numbers of black Americans in control cohorts probably would have prevented these misclassifications. We identified methodologic shortcomings that contributed to these errors in the medical literature. Conclusions The misclassification of benign variants as pathogenic that we found in our study shows the need for sequencing the genomes of diverse populations, both in asymptomatic controls and the tested patient population. These results expand on current guidelines, which recommend the use of ancestry-matched controls to interpret variants. As additional populations of different ancestry backgrounds are sequenced, we expect variant reclassifications to increase, particularly for ancestry groups that have historically been less well studied. (Funded by the National Institutes of Health.).

Concepts: English American, Sequence, European American, Genetics, African American, United States, White American, Hypertrophic cardiomyopathy

173

We provide a novel method, DRISEE (duplicate read inferred sequencing error estimation), to assess sequencing quality (alternatively referred to as “noise” or “error”) within and/or between sequencing samples. DRISEE provides positional error estimates that can be used to inform read trimming within a sample. It also provides global (whole sample) error estimates that can be used to identify samples with high or varying levels of sequencing error that may confound downstream analyses, particularly in the case of studies that utilize data from multiple sequencing samples. For shotgun metagenomic data, we believe that DRISEE provides estimates of sequencing error that are more accurate and less constrained by technical limitations than existing methods that rely on reference genomes or the use of scores (e.g. Phred). Here, DRISEE is applied to (non amplicon) data sets from both the 454 and Illumina platforms. The DRISEE error estimate is obtained by analyzing sets of artifactual duplicate reads (ADRs), a known by-product of both sequencing platforms. We present DRISEE as an open-source, platform-independent method to assess sequencing error in shotgun metagenomic data, and utilize it to discover previously uncharacterized error in de novo sequence data from the 454 and Illumina sequencing platforms.

Concepts: Sequence, Error, Estimation, Sample, Scientific method, Full genome sequencing, Estimator, Java

172

Massively parallel high throughput sequencing technologies allow us to interrogate the microbial composition of biological samples at unprecedented resolution. The typical approach is to perform high-throughout sequencing of 16S rRNA genes, which are then taxonomically classified based on similarity to known sequences in existing databases. Current technologies cause a predicament though, because although they enable deep coverage of samples, they are limited in the length of sequence they can produce. As a result, high-throughout studies of microbial communities often do not sequence the entire 16S rRNA gene. The challenge is to obtain reliable representation of bacterial communities through taxonomic classification of short 16S rRNA gene sequences. In this study we explored properties of different study designs and developed specific recommendations for effective use of short-read sequencing technologies for the purpose of interrogating bacterial communities, with a focus on classification using naïve Bayesian classifiers. To assess precision and coverage of each design, we used a collection of ∼8,500 manually curated 16S rRNA gene sequences from cultured bacteria and a set of over one million bacterial 16S rRNA gene sequences retrieved from environmental samples, respectively. We also tested different configurations of taxonomic classification approaches using short read sequencing data, and provide recommendations for optimal choice of the relevant parameters. We conclude that with a judicious selection of the sequenced region and the corresponding choice of a suitable training set for taxonomic classification, it is possible to explore bacterial communities at great depth using current technologies, with only a minimal loss of taxonomic resolution.

Concepts: Taxonomy, Biotechnology, DNA, Sequence, Ribosomal RNA, Archaea, Bacteria, 16S ribosomal RNA

171

Emerging sequencing technologies allow common and rare variants to be systematically assayed across the human genome in many individuals. In order to improve variant detection and genotype calling, raw sequence data are typically examined across many individuals. Here, we describe a method for genotype calling in settings where sequence data are available for unrelated individuals and parent-offspring trios and show that modeling trio information can greatly increase the accuracy of inferred genotypes and haplotypes, especially on low to modest depth sequencing data. Our method considers both linkage disequilibrium (LD) patterns and the constraints imposed by family structure when assigning individual genotypes and haplotypes. Using simulations, we show that trios provide higher genotype calling accuracy across the frequency spectrum, both overall and at hard-to-call heterozygous sites. In addition, trios provide greatly improved phasing accuracy-improving the accuracy of downstream analyses (such as genotype imputation) that rely on phased haplotypes. To further evaluate our approach, we analyzed data on the first 508 individuals sequenced by the SardiNIA sequencing project. Our results show that our method reduces the genotyping error rate by 50% compared with analysis using existing methods that ignore family structure. We anticipate our method will facilitate genotype calling and haplotype inference for many ongoing sequencing projects.

Concepts: Zygosity, Sequence, Human genome, Linkage disequilibrium, Gene, DNA, Haplotype, Genetics

171

Analysis of microbial communities by high-throughput pyrosequencing of SSU rRNA gene PCR amplicons has transformed microbial ecology research and led to the observation that many communities contain a diverse assortment of rare taxa-a phenomenon termed the Rare Biosphere. Multiple studies have investigated the effect of pyrosequencing read quality on operational taxonomic unit (OTU) richness for contrived communities, yet there is limited information on the fidelity of community structure estimates obtained through this approach. Given that PCR biases are widely recognized, and further unknown biases may arise from the sequencing process itself, a priori assumptions about the neutrality of the data generation process are at best unvalidated. Furthermore, post-sequencing quality control algorithms have not been explicitly evaluated for the accuracy of recovered representative sequences and its impact on downstream analyses, reducing useful discussion on pyrosequencing reads to their diversity and abundances. Here we report on community structures and sequences recovered for in vitro-simulated communities consisting of twenty 16S rRNA gene clones tiered at known proportions. PCR amplicon libraries of the V3-V4 and V6 hypervariable regions from the in vitro-simulated communities were sequenced using the Roche 454 GS FLX Titanium platform. Commonly used quality control protocols resulted in the formation of OTUs with >1% abundance composed entirely of erroneous sequences, while over-aggressive clustering approaches obfuscated real, expected OTUs. The pyrosequencing process itself did not appear to impose significant biases on overall community structure estimates, although the detection limit for rare taxa may be affected by PCR amplicon size and quality control approach employed. Meanwhile, PCR biases associated with the initial amplicon generation may impose greater distortions in the observed community structure.

Concepts: Archaea, Sequence, DNA sequencing, Scientific method, 30S, Biotechnology, Ribosomal RNA, 16S ribosomal RNA

170

SUMMARY: Two methods to add unaligned sequences into an existing multiple sequence alignment have been implemented as the “–add” and “–addfragments” options in the MAFFT package. The former option is a basic one and applicable only to full-length sequences, while the latter option is applicable even when the unaligned sequences are short and fragmentary. These methods internally infer the phylogenetic relationship among the sequences in the existing alignment, as well as the phylogenetic positions of unaligned sequences. Benchmarks based on two independent simulations consistently suggest that the “–addfragments” option outperforms recent methods, PaPaRa and PAGAN, in accuracy for difficult problems and that these three methods appropriately handle easy problems. AVAILABILITY: http://mafft.cbrc.jp/alignment/software/ CONTACT: katoh@ifrec.osaka-u.ac.jp SUPPLEMENTARY INFORMATION: Available at Bioinformatics online.

Concepts: Sequence, Multiple sequence alignment, DNA, Sequence alignment, Clustal, Phylogenetic tree, Bioinformatics, Computational phylogenetics