High-throughput technologies are widely used, for example to assay genetic variants, gene and protein expression, and epigenetic modifications. One often overlooked complication with such studies is ...batch effects, which occur because measurements are affected by laboratory conditions, reagent lots and personnel differences. This becomes a major problem when batch effects are correlated with an outcome of interest and lead to incorrect conclusions. Using both published studies and our own analyses, we argue that batch effects (as well as other technical and biological artefacts) are widespread and critical to address. We review experimental and computational approaches for doing so.
Abstract
With the avalanche of biological sequences generated in the post-genomic age, one of the most challenging problems is how to computationally analyze their structures and functions. Machine ...learning techniques are playing key roles in this field. Typically, predictors based on machine learning techniques contain three main steps: feature extraction, predictor construction and performance evaluation. Although several Web servers and stand-alone tools have been developed to facilitate the biological sequence analysis, they only focus on individual step. In this regard, in this study a powerful Web server called BioSeq-Analysis (http://bioinformatics.hitsz.edu.cn/BioSeq-Analysis/) has been proposed to automatically complete the three main steps for constructing a predictor. The user only needs to upload the benchmark data set. BioSeq-Analysis can generate the optimized predictor based on the benchmark data set, and the performance measures can be reported as well. Furthermore, to maximize user’s convenience, its stand-alone program was also released, which can be downloaded from http://bioinformatics.hitsz.edu.cn/BioSeq-Analysis/download/, and can be directly run on Windows, Linux and UNIX. Applied to three sequence analysis tasks, experimental results showed that the predictors generated by BioSeq-Analysis even outperformed some state-of-the-art methods. It is anticipated that BioSeq-Analysis will become a useful tool for biological sequence analysis.
The rapid evolution of 454 GS-FLX sequencing technology has not been accompanied by a reassessment of the quality and accuracy of the sequences obtained. Current strategies for decision-making and ...error-correction are based on an initial analysis by Huse et al. in 2007, for the older GS20 system based on experimental sequences. We analyze here the quality of 454 sequencing data and identify factors playing a role in sequencing error, through the use of an extensive dataset for Roche control DNA fragments.
We obtained a mean error rate for 454 sequences of 1.07%. More importantly, the error rate is not randomly distributed; it occasionally rose to more than 50% in certain positions, and its distribution was linked to several experimental variables. The main factors related to error are the presence of homopolymers, position in the sequence, size of the sequence and spatial localization in PT plates for insertion and deletion errors. These factors can be described by considering seven variables. No single variable can account for the error rate distribution, but most of the variation is explained by the combination of all seven variables.
The pattern identified here calls for the use of internal controls and error-correcting base callers, to correct for errors, when available (e.g. when sequencing amplicons). For shotgun libraries, the use of both sequencing primers and deep coverage, combined with the use of random sequencing primer sites should partly compensate for even high error rates, although it may prove more difficult than previous thought to distinguish between low-frequency alleles and errors.
The National Center for Biotechnology Information (NCBI) Reference Sequence (RefSeq) database is a collection of genomic, transcript and protein sequence records. These records are selected and ...curated from public sequence archives and represent a significant reduction in redundancy compared to the volume of data archived by the International Nucleotide Sequence Database Collaboration. The database includes over 16 000 organisms, 2.4 × 106 genomic records, 13 × 106 proteins and 2 × 106 RNA records spanning prokaryotes, eukaryotes and viruses (RefSeq release 49, September 2011). The RefSeq database is maintained by a combined approach of automated analyses, collaboration and manual curation to generate an up-to-date representation of the sequence, its features, names and cross-links to related sources of information. We report here on recent growth, the status of curating the human RefSeq data set, more extensive feature annotation and current policy for eukaryotic genome annotation via the NCBI annotation pipeline. More information about the resource is available online (see http://www.ncbi.nlm.nih.gov/RefSeq/).
Microorganisms are ubiquitous in the biosphere, playing a crucial role in both biogeochemistry of the planet and human health. However, identifying these microorganisms and defining their function ...are challenging. Widely used approaches in comparative metagenomics, 16S amplicon sequencing and whole genome shotgun sequencing (WGS), have provided access to DNA sequencing analysis to identify microorganisms and evaluate diversity and abundance in various environments. However, advances in parallel high-throughput DNA sequencing in the past decade have introduced major hurdles, namely standardization of methods, data storage, reproducible interoperability of results, and data sharing. The National Ecological Observatory Network (NEON), established by the National Science Foundation, enables all researchers to address queries on a regional to continental scale around a variety of environmental challenges and provide high-quality, integrated, and standardized data from field sites across the U.S. As the amount of metagenomic data continues to grow, standardized procedures that allow results across projects to be assessed and compared is becoming increasingly important in the field of metagenomics. We demonstrate the feasibility of using publicly available NEON soil metagenomic sequencing datasets in combination with open access Metagenomics Rapid Annotation using the Subsystem Technology (MG-RAST) server to illustrate advantages of WGS compared to 16S amplicon sequencing. Four WGS and four 16S amplicon sequence datasets, from surface soil samples prepared by NEON investigators, were selected for comparison, using standardized protocols collected at the same locations in Colorado between April-July 2014. The dominant bacterial phyla detected across samples agreed between sequencing methodologies. However, WGS yielded greater microbial resolution, increased accuracy, and allowed identification of more genera of bacteria, archaea, viruses, and eukaryota, and putative functional genes that would have gone undetected using 16S amplicon sequencing. NEON open data will be useful for future studies characterizing and quantifying complex ecological processes associated with changing aquatic and terrestrial ecosystems.
Repetitive DNA sequences are abundant in a broad range of species, from bacteria to mammals, and they cover nearly half of the human genome. Repeats have always presented technical challenges for ...sequence alignment and assembly programs. Next-generation sequencing projects, with their short read lengths and high data volumes, have made these challenges more difficult. From a computational perspective, repeats create ambiguities in alignment and assembly, which, in turn, can produce biases and errors when interpreting results. Simply ignoring repeats is not an option, as this creates problems of its own and may mean that important biological phenomena are missed. We discuss the computational problems surrounding repeats and describe strategies used by current bioinformatics systems to solve them.
Abstract
As the first web server to analyze various biological sequences at sequence level based on machine learning approaches, many powerful predictors in the field of computational biology have ...been developed with the assistance of the BioSeq-Analysis. However, the BioSeq-Analysis can be only applied to the sequence-level analysis tasks, preventing its applications to the residue-level analysis tasks, and an intelligent tool that is able to automatically generate various predictors for biological sequence analysis at both residue level and sequence level is highly desired. In this regard, we decided to publish an important updated server covering a total of 26 features at the residue level and 90 features at the sequence level called BioSeq-Analysis2.0 (http://bliulab.net/BioSeq-Analysis2.0/), by which the users only need to upload the benchmark dataset, and the BioSeq-Analysis2.0 can generate the predictors for both residue-level analysis and sequence-level analysis tasks. Furthermore, the corresponding stand-alone tool was also provided, which can be downloaded from http://bliulab.net/BioSeq-Analysis2.0/download/. To the best of our knowledge, the BioSeq-Analysis2.0 is the first tool for generating predictors for biological sequence analysis tasks at residue level. Specifically, the experimental results indicated that the predictors developed by BioSeq-Analysis2.0 can achieve comparable or even better performance than the existing state-of-the-art predictors.
Demand has never been greater for revolutionary technologies that deliver fast, inexpensive and accurate genome information. This challenge has catalysed the development of next-generation sequencing ...(NGS) technologies. The inexpensive production of large volumes of sequence data is the primary advantage over conventional methods. Here, I present a technical review of template preparation, sequencing and imaging, genome alignment and assembly approaches, and recent advances in current and near-term commercially available NGS instruments. I also outline the broad range of applications for NGS technologies, in addition to providing guidelines for platform selection to address biological questions of interest.
Two complementary approaches, both using next-generation sequencing, have successfully tackled the scale and the complexity of mammalian transcriptomes. The techniques reveal unprecedented detail and ...allow better quantification.
Next-generation massively parallel DNA sequencing technologies provide ultrahigh throughput at a substantially lower unit data cost; however, the data are very short read length sequences, making de ...novo assembly extremely challenging. Here, we describe a novel method for de novo assembly of large genomes from short read sequences. We successfully assembled both the Asian and African human genome sequences, achieving an N50 contig size of 7.4 and 5.9 kilobases (kb) and scaffold of 446.3 and 61.9 kb, respectively. The development of this de novo short read assembly method creates new opportunities for building reference sequences and carrying out accurate analyses of unexplored genomes in a cost-effective way.
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