DNA sequencing continues to decrease in cost with the Illumina HiSeq2000 generating up to 600 Gb of paired-end 100 base reads in a ten-day run. Here we present a protocol for community amplicon sequencing on the HiSeq2000 and MiSeq Illumina platforms, and apply that protocol to sequence 24 microbial communities from host-associated and free-living environments. A critical question as more sequencing platforms become available is whether biological conclusions derived on one platform are consistent with what would be derived on a different platform. We show that the protocol developed for these instruments successfully recaptures known biological results, and additionally that biological conclusions are consistent across sequencing platforms (the HiSeq2000 versus the MiSeq) and across the sequenced regions of amplicons.

DNA sequence information underpins genetic research, enabling discoveries of important biological or medical benefit. Sequencing projects have traditionally employed long (400–800 bp) 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 intra-species genetic variation. 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 >30x average depth of paired 35-base reads. We characterise four million SNPs and four hundred thousand structural variants, many of which are previously unknown. Our approach is effective for accurate, rapid and economical whole genome re-sequencing and many other biomedical applications.

All cancers carry somatic mutations. A subset of these somatic alterations, termed driver mutations, confer selective growth advantage and are implicated in cancer development, whereas the remainder are passengers. Here we have sequenced the genomes of a malignant melanoma and a lymphoblastoid cell line from the same person, providing the first comprehensive catalogue of somatic mutations from an individual cancer. The catalogue provides remarkable insights into the forces that have shaped this cancer genome. The dominant mutational signature reflects DNA damage due to ultraviolet light exposure, a known risk factor for malignant melanoma, whereas the uneven distribution of mutations across the genome, with a lower prevalence in gene footprints, indicates that DNA repair has been preferentially deployed towards transcribed regions. The results illustrate the power of a cancer genome sequence to reveal traces of the DNA damage, repair, mutation and selection processes that were operative years before the cancer became symptomatic.

Human T-lymphotropic virus type 1 (HTLV-1) persists by driving clonal proliferation of infected T lymphocytes. A high proviral load predisposes to HTLV-1-associated diseases. Yet the reasons for the variation within and between persons in the abundance of HTLV-1-infected clones remain unknown. We devised a highthroughput protocol to map the genomic location and quantify the abundance of > 91 000 unique insertion sites of the provirus from 61 HTLV-1 ؉ persons and > 2100 sites from in vitro infection. We show that a typical HTLV-1-infected host carries between 500 and 5000 unique insertion sites. We demonstrate that negative selection dominates during chronic infection, favoring establishment of proviruses integrated in transcriptionally silenced DNA: this selection is significantly stronger in asymptomatic carriers. We define a parameter, the oligoclonality index, to quantify clonality. The high proviral load characteristic of HTLV-1- IntroductionHuman T-lymphotropic virus type 1 (HTLV-1) causes adult T-cell leukemia-lymphoma (ATLL), HTLV-1-associated myelopathy/ tropical spastic paraparesis (HAM/TSP), uveitis, and infective dermatitis. It is estimated that 15 to 20 million persons live with HTLV-1 infection worldwide. A small proportion (up to 7%, depending on the area) of HTLV-1-infected persons develop disease, whereas the majority remain asymptomatic carriers (ACs). Infection occurs via breastfeeding, transfusion of infected cellular blood products, or sexual intercourse. Symptoms appear after a long period (years or decades) of clinical latency. 1 The HTLV-1 proviral load (PVL) remains stable within each infected person and correlates with the outcome of infection. However, the PVL varies widely among infected people, even within a particular diagnostic group. [2][3][4] The sequence of HTLV-1 is also stable within a person, 5,6 indicating that the PVL is maintained in vivo mainly by mitosis of infected cells during the chronic phase of the infection. This interpretation is supported by the observation that individual clones of infected cells can persist in patients for several years. 7-9 Thus, it has been hypothesized that infectious transmission of HTLV-1 is important early in infection across the virologic synapse, 10 whereas mitotic replication is responsible for maintaining proviral load once a persistent infection has been established and reached an equilibrium with the immune response. 11 In approximately 5% of infected people, persistent clonal proliferation culminates in malignant transformation in the disease ATLL. 7,8 The leukemic clones carry generally one (complete or defective) provirus per cell. [12][13][14] There has been a longstanding debate on the question of whether HTLV-1 is latent or persistently expressed in vivo. Persistent expression is strongly suggested by the extensive evidence that the strong, chronically activated cytotoxic T lymphocyte (CTL) response to HTLV-1 limits the proviral load and reduces the risk of HAM/TSP. 11 Furthermore, there is both experimental evidence 15 and th...

SummaryThe Tasmanian devil (Sarcophilus harrisii), the largest marsupial carnivore, is endangered due to a transmissible facial cancer spread by direct transfer of living cancer cells through biting. Here we describe the sequencing, assembly, and annotation of the Tasmanian devil genome and whole-genome sequences for two geographically distant subclones of the cancer. Genomic analysis suggests that the cancer first arose from a female Tasmanian devil and that the clone has subsequently genetically diverged during its spread across Tasmania. The devil cancer genome contains more than 17,000 somatic base substitution mutations and bears the imprint of a distinct mutational process. Genotyping of somatic mutations in 104 geographically and temporally distributed Tasmanian devil tumors reveals the pattern of evolution and spread of this parasitic clonal lineage, with evidence of a selective sweep in one geographical area and persistence of parallel lineages in other populations.PaperClip