Since the completion of the human genome project in 2003, extraordinary progress has been made in genome sequencing technologies, which has led to a decreased cost per megabase and an increase in the number and diversity of sequenced genomes. An astonishing complexity of genome architecture has been revealed, bringing these sequencing technologies to even greater advancements. Some approaches maximize the number of bases sequenced in the least amount of time, generating a wealth of data that can be used to understand increasingly complex phenotypes. Alternatively, other approaches now aim to sequence longer contiguous pieces of DNA, which are essential for resolving structurally complex regions. These and other strategies are providing researchers and clinicians a variety of tools to probe genomes in greater depth, leading to an enhanced understanding of how genome sequence variants underlie phenotype and disease.

Coming of age: ten years of next-generation sequencing technologies

Relation between the gut microbiota and human health is being increasingly recognised It is now well established that a healthy gut flora is largely responsible for overall health of the host The normal human gut microbiota comprises of two major phyla, namely Bacteroidetes and Firmicutes Though the gut microbiota in an infant appears haphazard, it starts resembling the adult flora by the age of 3 years Nevertheless, there exist temporal and spatial variations in the microbial distribution from esophagus to the rectum all along the individual's life span Developments in genome sequencing technologies and bioinformatics have now enabled scientists to study these microorganisms and their function and microbe-host interactions in an elaborate manner both in health and disease The normal gut microbiota imparts specific function in host nutrient metabolism, xenobiotic and drug metabolism, maintenance of structural integrity of the gut mucosal barrier, immunomodulation, and protection against pathogens Several factors play a role in shaping the normal gut microbiota They include (1) the mode of delivery (vaginal or caesarean); (2) diet during infancy (breast milk or formula feeds) and adulthood (vegan based or meat based); and (3) use of antibiotics or antibiotic like molecules that are derived from the environment or the gut commensal community A major concern of antibiotic use is the long-term alteration of the normal healthy gut microbiota and horizontal transfer of resistance genes that could result in reservoir of organisms with a multidrug resistant gene pool

Role of the normal gut microbiota

PacBio Sequencing and Its Applications.

The recent advent of methods for high-throughput single-cell molecular profiling has catalyzed a growing sense in the scientific community that the time is ripe to complete the 150-year-old effort to identify all cell types in the human body. The Human Cell Atlas Project is an international collaborative effort that aims to define all human cell types in terms of distinctive molecular profiles (such as gene expression profiles) and to connect this information with classical cellular descriptions (such as location and morphology). An open comprehensive reference map of the molecular state of cells in healthy human tissues would propel the systematic study of physiological states, developmental trajectories, regulatory circuitry and interactions of cells, and also provide a framework for understanding cellular dysregulation in human disease. Here we describe the idea, its potential utility, early proofs-of-concept, and some design considerations for the Human Cell Atlas, including a commitment to open data, code, and community.

The Human Cell Atlas

Long-read technologies are overcoming early limitations in accuracy and throughput, broadening their application domains in genomics. Dedicated analysis tools that take into account the characteristics of long-read data are thus required, but the fast pace of development of such tools can be overwhelming. To assist in the design and analysis of long-read sequencing projects, we review the current landscape of available tools and present an online interactive database, long-read-tools.org, to facilitate their browsing. We further focus on the principles of error correction, base modification detection, and long-read transcriptomics analysis and highlight the challenges that remain.

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Opportunities and challenges in long-read sequencing data analysis.

Next generation sequencing (NGS) technology has revolutionized genomic and genetic research. The pace of change in this area is rapid with three major new sequencing platforms having been released in 2011: Ion Torrent’s PGM, Pacific Biosciences’ RS and the Illumina MiSeq. Here we compare the results obtained with those platforms to the performance of the Illumina HiSeq, the current market leader. In order to compare these platforms, and get sufficient coverage depth to allow meaningful analysis, we have sequenced a set of 4 microbial genomes with mean GC content ranging from 19.3 to 67.7%. Together, these represent a comprehensive range of genome content. Here we report our analysis of that sequence data in terms of coverage distribution, bias, GC distribution, variant detection and accuracy. Sequence generated by Ion Torrent, MiSeq and Pacific Biosciences technologies displays near perfect coverage behaviour on GC-rich, neutral and moderately AT-rich genomes, but a profound bias was observed upon sequencing the extremely AT-rich genome of Plasmodium falciparum on the PGM, resulting in no coverage for approximately 30% of the genome. We analysed the ability to call variants from each platform and found that we could call slightly more variants from Ion Torrent data compared to MiSeq data, but at the expense of a higher false positive rate. Variant calling from Pacific Biosciences data was possible but higher coverage depth was required. Context specific errors were observed in both PGM and MiSeq data, but not in that from the Pacific Biosciences platform. All three fast turnaround sequencers evaluated here were able to generate usable sequence. However there are key differences between the quality of that data and the applications it will support.

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A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers.

G&T-seq offers robust full-length transcript and whole-genome sequencing simultaneously from a single cell.

G&T-seq: parallel sequencing of single-cell genomes and transcriptomes

A minor but significant fraction of samples subjected to next-generation sequencing methods are either mixed-up or cross-contaminated. These events can lead to false or inconclusive results. We have therefore developed SASI-Seq; a process whereby a set of uniquely barcoded DNA fragments are added to samples destined for sequencing. From the final sequencing data, one can verify that all the reads derive from the original sample(s) and not from contaminants or other samples. By adding a mixture of three uniquely barcoded amplicons, of different sizes spanning the range of insert sizes one would normally use for Illumina sequencing, at a spike-in level of approximately 0.1%, we demonstrate that these fragments remain intimately associated with the sample. They can be detected following even the tightest size selection regimes or exome enrichment and can report the occurrence of sample mix-ups and cross-contamination. As a consequence of this work, we have designed a set of 384 eleven-base Illumina barcode sequences that are at least 5 changes apart from each other, allowing for single-error correction and very low levels of barcode misallocation due to sequencing error. SASI-Seq is a simple, inexpensive and flexible tool that enables sample assurance, allows deconvolution of sample mix-ups and reports levels of cross-contamination between samples throughout NGS workflows.

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SASI-Seq: sample assurance Spike-Ins, and highly differentiating 384 barcoding for Illumina sequencing

Studies on DNA from pathogenic organisms, within clinical samples, are often complicated by the presence of large amounts of host, e.g., human DNA. Isolation of pathogen DNA from these samples would improve the efficiency of next-generation sequencing (NGS) and pathogen identification. Here we describe a solution-based hybridisation method for isolation of pathogen DNA from a mixed population. This straightforward and inexpensive technique uses probes made from whole-genome DNA and off-the-shelf reagents. In this study, Escherichia coli DNA was successfully enriched from a mixture of E.coli and human DNA. After enrichment, genome coverage following NGS was significantly higher and the evenness of coverage and GC content were unaffected. This technique was also applied to samples containing a mixture of human and Plasmodium falciparum DNA. The P.falciparum genome is particularly difficult to sequence due to its high AT content (80.6%) and repetitive nature. Post enrichment, a bias in the recovered DNA was observed, with a poorer representation of the AT-rich non-coding regions. This uneven coverage was also observed in pre-enrichment samples, but to a lesser degree. Despite the coverage bias in enriched samples, SNP (single-nucleotide polymorphism) calling in coding regions was unaffected and the majority of samples had over 90% of their coding region covered at 5× depth. This technique shows significant promise as an effective method to enrich pathogen DNA from samples with heavy human contamination, particularly when applied to GC-neutral genomes.

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Miriam Smith

Papers

A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers.

G&T-seq: parallel sequencing of single-cell genomes and transcriptomes

SASI-Seq: sample assurance Spike-Ins, and highly differentiating 384 barcoding for Illumina sequencing

An In-Solution Hybridisation Method for the Isolation of Pathogen DNA from Human DNA-rich Clinical Samples for Analysis by NGS