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DNA sequence information underpins genetic research, enabling discoveries of important biological or medical benefit. Sequencing projects have traditionally used long (400–800 base pair) reads, but the existence of reference sequences for the human and many other genomes makes it possible to develop new, fast approaches to re-sequencing, whereby shorter reads are compared to a reference to identify intraspecies genetic variation. Here we report an approach that generates several billion bases of accurate nucleotide sequence per experiment at low cost. Single molecules of DNA are attached to a flat surface, amplified in situ and used as templates for synthetic sequencing with fluorescent reversible terminator deoxyribonucleotides. Images of the surface are analysed to generate high-quality sequence. We demonstrate application of this approach to human genome sequencing on flow-sorted X chromosomes and then scale the approach to determine the genome sequence of a male Yoruba from Ibadan, Nigeria. We build an accurate consensus sequence from >30× average depth of paired 35-base reads. We characterize four million single-nucleotide polymorphisms and four hundred thousand structural variants, many of which were previously unknown. Our approach is effective for accurate, rapid and economical whole-genome re-sequencing and many other biomedical applications. [ABSTRACT FROM AUTHOR]

The simultaneous sequencing of a single cell's genome and transcriptome offers a powerful means to dissect genetic variation and its effect on gene expression. Here we describe G&T-seq, a method for separating and sequencing genomic DNA and full-length mRNA from single cells. By applying G&T-seq to over 220 single cells from mice and humans, we discovered cellular properties that could not be inferred from DNA or RNA sequencing alone. [ABSTRACT FROM AUTHOR]

Background 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. Results 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. Conclusion 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. [ABSTRACT FROM AUTHOR]

Background: 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. Results: 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. Conclusions: 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. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented which discusses the identification of optimal enzymes for amplifying high complexity mixtures of DNA fragments.