Clinical and pathological impact of VHL, PBRM1, BAP1, SETD2, KDM6A, and JARID1c in clear cell renal cell carcinoma.

VHL is mutated in the majority of patients with clear cell renal cell carcinoma (ccRCC), with conflicting clinical relevance. Recent studies have identified recurrent mutations in histone modifying and chromatin remodeling genes, including BAP1, PBRM1, SETD2, KDM6A, and JARID1c. Current evidence suggests that BAP1 mutations are associated with aggressive disease. The clinical significance of the remaining genes is unknown. In this study, targeted sequencing of VHL and JARID1c (entire genes) and coding regions of BAP1, PBRM1, SETD2, and KDM6A was performed on 132 ccRCCs and matched normal tissues. Associations between mutations and clinical and pathological outcomes were interrogated. Inactivation of VHL (coding mutation or promoter methylation) was seen in 75% of ccRCCs. Somatic noncoding VHL alterations were identified in 29% of ccRCCs and may be associated with improved overall survival. BAP1 (11%), PBRM1 (33%), SETD2 (16%), JARID1c (4%), and KDM6A (3%) mutations were identified. BAP1-mutated tumors were associated with metastatic disease at presentation (P = 0.023), advanced clinical stage (P = 0.042) and a trend towards shorter recurrence free survival (P = 0.059) when compared with tumors exclusively mutated for PBRM1. Our results support those of recent publications pointing towards a role for BAP1 and PBRM1 mutations in risk stratifying ccRCCs. Further investigation of noncoding alterations in VHL is warranted.

Reflex: intramolecular barcoding of long-range PCR products for sequencing multiple pooled DNAs.

We present an intramolecular reaction, Reflex™, to derive shorter, sequencer-ready, daughter polymerase chain reaction products from a pooled population of barcoded long-range polymerase chain reaction products, whilst still preserving the cognate DNA barcodes. Our Reflex workflow needs only a small number of primer extension steps to rapidly enable uniform sequence coverage of long contiguous sequence targets in large numbers of samples at low cost on desktop next-generation sequencers.

Stops making sense: translational trade-offs and stop codon reassignment.

BACKGROUND: Efficient gene expression involves a trade-off between (i) premature termination of protein synthesis; and (ii) readthrough, where the ribosome fails to dissociate at the terminal stop. Sense codons that are similar in sequence to stop codons are more susceptible to nonsense mutation, and are also likely to be more susceptible to transcriptional or translational errors causing premature termination. We therefore expect this trade-off to be influenced by the number of stop codons in the genetic code. Although genetic codes are highly constrained, stop codon number appears to be their most volatile feature. RESULTS: In the human genome, codons readily mutable to stops are underrepresented in coding sequences. We construct a simple mathematical model based on the relative likelihoods of premature termination and readthrough. When readthrough occurs, the resultant protein has a tail of amino acid residues incorrectly added to the C-terminus. Our results depend strongly on the number of stop codons in the genetic code. When the code has more stop codons, premature termination is relatively more likely, particularly for longer genes. When the code has fewer stop codons, the length of the tail added by readthrough will, on average, be longer, and thus more deleterious. Comparative analysis of taxa with a range of stop codon numbers suggests that genomes whose code includes more stop codons have shorter coding sequences. CONCLUSIONS: We suggest that the differing trade-offs presented by alternative genetic codes may result in differences in genome structure. More speculatively, multiple stop codons may mitigate readthrough, counteracting the disadvantage of a higher rate of nonsense mutation. This could help explain the puzzling overrepresentation of stop codons in the canonical genetic code and most variants.

A method for counting PCR template molecules with application to next-generation sequencing.

Amplification by polymerase chain reaction is often used in the preparation of template DNA molecules for next-generation sequencing. Amplification increases the number of available molecules for sequencing but changes the representation of the template molecules in the amplified product and introduces random errors. Such changes in representation hinder applications requiring accurate quantification of template molecules, such as allele calling or estimation of microbial diversity. We present a simple method to count the number of template molecules using degenerate bases and show that it improves genotyping accuracy and removes noise from PCR amplification. This method can be easily added to existing DNA library preparation techniques and can improve the accuracy of variant calling.

The origin of tobacco's T genome is traced to a particular lineage within Nicotiana tomentosiformis (Solanaceae).

Nicotiana tabacum (tobacco) is a natural allotetraploid. The maternal genome donor is not controversial and is probably derived from an ancestor of N. sylvestris. The paternal, T-genome donor has been less clear, with N. tomentosiformis, N. otophora, or an introgression hybrid proposed. Here we provide evidence that the T genome of N. tabacum is derived from a particular lineage of N. tomentosiformis. We show that the repetitive sequences of geminiviral origin, GRD53 and GRD3, are present in the genomes of N. tabacum cultivars, a tobacco cell suspension culture TBY-2, and N. tomentosiformis ac. NIC 479/84. Surprisingly, they are not present in another three varieties of N. tomentosiformis. A detailed cytogenetic analysis also revealed that N. tomentosiformis ac. NIC 479/84 most closely resembles the N. tabacum T genome in the location of other tandem repetitive sequences. Thus, tobacco formed after divergence within N. tomentosiformis, and the spectrum of potential donors of the paternal genome can be narrowed to a genotype of N. tomentosiformis characterized by the presence of GRD53 and GRD3 repeats. It is clear that future paternity studies in tobacco should use N. tomentosiformis ac. NIC 479/84 rather than any other accession.