Accurate whole human genome sequencing using reversible terminator chemistry.

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 >30x 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.

Strelka: accurate somatic small-variant calling from sequenced tumor-normal sample pairs.

MOTIVATION: Whole genome and exome sequencing of matched tumor-normal sample pairs is becoming routine in cancer research. The consequent increased demand for somatic variant analysis of paired samples requires methods specialized to model this problem so as to sensitively call variants at any practical level of tumor impurity. RESULTS: We describe Strelka, a method for somatic SNV and small indel detection from sequencing data of matched tumor-normal samples. The method uses a novel Bayesian approach which represents continuous allele frequencies for both tumor and normal samples, while leveraging the expected genotype structure of the normal. This is achieved by representing the normal sample as a mixture of germline variation with noise, and representing the tumor sample as a mixture of the normal sample with somatic variation. A natural consequence of the model structure is that sensitivity can be maintained at high tumor impurity without requiring purity estimates. We demonstrate that the method has superior accuracy and sensitivity on impure samples compared with approaches based on either diploid genotype likelihoods or general allele-frequency tests. AVAILABILITY: The Strelka workflow source code is available at ftp://strelka@ftp.illumina.com/. CONTACT: csaunders@illumina.com

Transmucosal gastric leak induced by proton pump inhibitors.

Despite their remarkable safety profile and lack of clinical side effects, proton pump inhibitors (PPIs) induce a transmucosal gastric leak to non-electrolyte probes of various sizes. The ex vivo addition of PPIs to isolated rat gastric corpus increases transmucosal permeability in a dose-dependent manner, which corresponds with PPIs' dose-dependent inhibition of acid secretion. Upon the addition of omeprazole, lansoprazole, or esomeprazole, a small decrease in transepithelial resistance and the concomitant stimulation of short circuit current was observed. Additionally, transepithelial flux of (14)C-[D]-mannitol (MW 182.17) across the gastric mucosa increased by a mean of 68% immediately following the addition of 200 microM omeprazole. This flux increase was bidirectional. Omeprazole also increased the paracellular permeability to larger radiolabeled probes, including (14)C-sucrose (MW 342.3) and (14)C-polyethylene glycol (MW 4,000) by 118% and 350%, respectively. However, the flux of still larger probes, 10,000 and 70,000 MW dextrans, was not increased. Because PPIs are so widely used and are assumed to be innocuous, this transmucosal gastric leak must be further investigated, as it may carry considerable biomedical implications.

Absence of Na+/sugar cotransport activity in Barrett's metaplasia.

AIM: To evaluate the presence of Na+-dependent, active, sugar transport in Barrett's epithelia as an intestinal biomarker, based on the well-documented, morphological intestinal phenotype of Barrett's esophagus (BE). METHODS: We examined uptake of the nonmeta-bolizable glucose analogue, alpha-methyl-D-glucoside (AMG), a substrate for the entire sodium glucose cotransporter (SGLT) family of transport proteins. During upper endoscopy, patients with BE or with uncomplicated gastroesophageal reflux disease (GERD) allowed for duodenal, gastric fundic, and esophageal mucosal biopsies to be taken. Biopsies were incubated in bicarbonate-buffered saline (KRB) containing 0.1 mmol/L 14C-AMG for 60 min at 20 centigrade. Characterized by abundant SGLT, duodenum served as a positive control while gastric fundus and normal esophagus, known to lack SGLT, served as negative controls. RESULTS: Duodenal biopsies accumulated 249.84+/-35.49 (SEM) picomoles AMG/microg DNA (n=12), gastric fundus biopsies 36.20+/-6.62 (n=12), normal esophagus 12.10+/-0.59 (n=3) and Barrett's metaplasia 29.79+/-5.77 (n=8). There was a statistical difference (P<0.01) between biopsies from duodenum and each other biopsy site but there was no statistically significant difference between normal esophagus and BE biopsies. 0.5 mmol/L phlorizin (PZ) inhibited AMG uptake into duodenal mucosa by over 89%, but had no significant effect on AMG uptake into gastric fundus, normal esophagus, or Barrett's tissue. In the absence of Na+ (all Na+ salts replaced by Li+ salts), AMG uptake in duodenum was decreased by over 90%, while uptake into gastric, esophageal or Barrett's tissue was statistically unaffected. CONCLUSION: Despite the intestinal enterocyte phenotype of BE, Na+-dependent, sugar transport activity is not present in these cells.

Proton pump inhibitors: actions and reactions.

Proton pump inhibitors are the second most commonly prescribed drug class in the United States. The increased utilization of PPIs parallels the rising incidence of reflux disease. Owing to their clinical efficacy and relative lack of tachyphylaxis, PPIs have largely displaced H-2 receptor antagonists in the treatment of acid peptic disorders. The elevation of intragastric pH and subsequent alterations of gastric physiology induced by PPIs may yield undesired effects within the upper GI tract. The ubiquity of the various types of H(+), K(+)-ATPase could also contribute to non-gastric effects. PPIs may influence physiology in other ways, such as inducing transepithelial leak.

Genomic diversity among drug sensitive and multidrug resistant isolates of Mycobacterium tuberculosis with identical DNA fingerprints.

BACKGROUND: Mycobacterium tuberculosis complex (MTBC), the causative agent of tuberculosis (TB), is characterized by low sequence diversity making this bacterium one of the classical examples of a genetically monomorphic pathogen. Because of this limited DNA sequence variation, routine genotyping of clinical MTBC isolates for epidemiological purposes relies on highly discriminatory DNA fingerprinting methods based on mobile and repetitive genetic elements. According to the standard view, isolates exhibiting the same fingerprinting pattern are considered direct progeny of the same bacterial clone, and most likely reflect ongoing transmission or disease relapse within individual patients. METHODOLOGY/PRINCIPAL FINDINGS: Here we further investigated this assumption and used massively parallel whole-genome sequencing to compare one drug-susceptible (K-1) and one multidrug resistant (MDR) isolate (K-2) of a rapidly spreading M. tuberculosis Beijing genotype clone from a high incidence region (Karakalpakstan, Uzbekistan). Both isolates shared the same IS6110 RFLP pattern and the same allele at 23 out of 24 MIRU-VNTR loci. We generated 23.9 million (K-1) and 33.0 million (K-2) paired 50 bp purity filtered reads corresponding to a mean coverage of 483.5 fold and 656.1 fold respectively. Compared with the laboratory strain H37Rv both Beijing isolates shared 1,209 SNPs. The two Beijing isolates differed by 130 SNPs and one large deletion. The susceptible isolate had 55 specific SNPs, while the MDR variant had 75 specific SNPs, including the five known resistance-conferring mutations. CONCLUSIONS: Our results suggest that M. tuberculosis isolates exhibiting identical DNA fingerprinting patterns can harbour substantial genomic diversity. Because this heterogeneity is not captured by traditional genotyping of MTBC, some aspects of the transmission dynamics of tuberculosis could be missed or misinterpreted. Furthermore, a valid differentiation between disease relapse and exogenous reinfection might be impossible using standard genotyping tools if the overall diversity of circulating clones is limited. These findings have important implications for clinical trials of new anti-tuberculosis drugs.