Piotr A. Mieczkowski

MapSplice: Accurate mapping of RNA-seq reads for splice junction discovery

The accurate mapping of reads that span splice junctions is a critical component of all analytic techniques that work with RNA-seq data. We introduce a second generation splice detection algorithm, MapSplice, whose focus is high sensitivity and specificity in the detection of splices as well as CPU and memory efficiency. MapSplice can be applied to both short (<75 bp) and long reads (≥75 bp). MapSplice is not dependent on splice site features or intron length, consequently it can detect novel canonical as well as non-canonical splices. MapSplice leverages the quality and diversity of read alignments of a given splice to increase accuracy. We demonstrate that MapSplice achieves higher sensitivity and specificity than TopHat and SpliceMap on a set of simulated RNA-seq data. Experimental studies also support the accuracy of the algorithm. Splice junctions derived from eight breast cancer RNA-seq datasets recapitulated the extensiveness of alternative splicing on a global level as well as the differences between molecular subtypes of breast cancer. These combined results indicate that MapSplice is a highly accurate algorithm for the alignment of RNA-seq reads to splice junctions. Software download URL: http://www.netlab.uky.edu/p/bioinfo/MapSplice.

A map of open chromatin in human pancreatic islets

Tissue-specific transcriptional regulation is central to human disease. To identify regulatory DNA active in human pancreatic islets, we profiled chromatin by formaldehyde-assisted isolation of regulatory elements coupled with high-throughput sequencing (FAIRE-seq). We identified ∼80,000 open chromatin sites. Comparison of FAIRE-seq data from islets to that from five non-islet cell lines revealed ∼3,300 physically linked clusters of islet-selective open chromatin sites, which typically encompassed single genes that have islet-specific expression. We mapped sequence variants to open chromatin sites and found that rs7903146, a TCF7L2 intronic variant strongly associated with type 2 diabetes, is located in islet-selective open chromatin. We found that human islet samples heterozygous for rs7903146 showed allelic imbalance in islet FAIRE signals and that the variant alters enhancer activity, indicating that genetic variation at this locus acts in cis with local chromatin and regulatory changes. These findings illuminate the tissue-specific organization of cis-regulatory elements and show that FAIRE-seq can guide the identification of regulatory variants underlying disease susceptibility.

Nucleosome Dynamics Define Transcriptional Enhancers

Chromatin plays a central role in eukaryotic gene regulation. We performed genome-wide mapping of epigenetically marked nucleosomes to determine their position both near transcription start sites and at distal regulatory elements, including enhancers. In prostate cancer cells, where androgen receptor binds primarily to enhancers, we found that androgen treatment dismisses a central nucleosome present at androgen receptor binding sites that is flanked by a pair of marked nucleosomes. A new quantitative model built on the behavior of such nucleosome pairs correctly identified regions bound by the regulators of the immediate androgen response, including androgen receptor and FOXA1. More importantly, this model also correctly predicted previously unidentified binding sites for other transcription factors present after prolonged androgen stimulation, including OCT1 and NKX3-1. Therefore, quantitative modeling of enhancer structure provides a powerful predictive method to infer the identity of transcription factors involved in cellular responses to specific stimuli.

Genetic heterogeneity of diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) is the most common form of lymphoma in adults. The disease exhibits a striking heterogeneity in gene expression profiles and clinical outcomes, but its genetic causes remain to be fully defined. Through whole genome and exome sequencing, we characterized the genetic diversity of DLBCL. In all, we sequenced 73 DLBCL primary tumors (34 with matched normal DNA). Separately, we sequenced the exomes of 21 DLBCL cell lines. We identified 322 DLBCL cancer genes that were recurrently mutated in primary DLBCLs. We identified recurrent mutations implicating a number of known and not previously identified genes and pathways in DLBCL including those related to chromatin modification (ARID1A and MEF2B), NF-κB (CARD11 and TNFAIP3), PI3 kinase (PIK3CD, PIK3R1, and MTOR), B-cell lineage (IRF8, POU2F2, and GNA13), and WNT signaling (WIF1). We also experimentally validated a mutation in PIK3CD, a gene not previously implicated in lymphomas. The patterns of mutation demonstrated a classic long tail distribution with substantial variation of mutated genes from patient to patient and also between published studies. Thus, our study reveals the tremendous genetic heterogeneity that underlies lymphomas and highlights the need for personalized medicine approaches to treating these patients.

Genome structure of a Saccharomyces cerevisiae strain widely used in bioethanol production

Bioethanol is a biofuel produced mainly from the fermentation of carbohydrates derived from agricultural feedstocks by the yeast Saccharomyces cerevisiae. One of the most widely adopted strains is PE-2, a heterothallic diploid naturally adapted to the sugar cane fermentation process used in Brazil. Here we report the molecular genetic analysis of a PE-2 derived diploid (JAY270), and the complete genome sequence of a haploid derivative (JAY291). The JAY270 genome is highly heterozygous (approximately 2 SNPs/kb) and has several structural polymorphisms between homologous chromosomes. These chromosomal rearrangements are confined to the peripheral regions of the chromosomes, with breakpoints within repetitive DNA sequences. Despite its complex karyotype, this diploid, when sporulated, had a high frequency of viable spores. Hybrid diploids formed by outcrossing with the laboratory strain S288c also displayed good spore viability. Thus, the rearrangements that exist near the ends of chromosomes do not impair meiosis, as they do not span regions that contain essential genes. This observation is consistent with a model in which the peripheral regions of chromosomes represent plastic domains of the genome that are free to recombine ectopically and experiment with alternative structures. We also explored features of the JAY270 and JAY291 genomes that help explain their high adaptation to industrial environments, exhibiting desirable phenotypes such as high ethanol and cell mass production and high temperature and oxidative stress tolerance. The genomic manipulation of such strains could enable the creation of a new generation of industrial organisms, ideally suited for use as delivery vehicles for future bioenergy technologies.

Practical innovations for high-throughput amplicon sequencing

We describe improvements for sequencing 16S ribosomal RNA (rRNA) amplicons, a cornerstone technique in metagenomics. Through unique tagging of template molecules before PCR, amplicon sequences can be mapped to their original templates to correct amplification bias and sequencing error with software we provide. PCR clamps block amplification of contaminating sequences from a eukaryotic host, thereby substantially enriching microbial sequences without introducing bias.

Double-strand breaks associated with repetitive DNA can reshape the genome

Ionizing radiation is an established source of chromosome aberrations (CAs). Although double-strand breaks (DSBs) are implicated in radiation-induced and other CAs, the underlying mechanisms are poorly understood. Here, we show that, although the vast majority of randomly induced DSBs in G2 diploid yeast cells are repaired efficiently through homologous recombination (HR) between sister chromatids or homologous chromosomes, ≈2% of all DSBs give rise to CAs. Complete molecular analysis of the genome revealed that nearly all of the CAs resulted from HR between nonallelic repetitive elements, primarily Ty retrotransposons. Nonhomologous end-joining (NHEJ) accounted for few, if any, of the CAs. We conclude that only those DSBs that fall at the 3–5% of the genome composed of repetitive DNA elements are efficient at generating rearrangements with dispersed small repeats across the genome, whereas DSBs in unique sequences are confined to recombinational repair between the large regions of homology contained in sister chromatids or homologous chromosomes. Because repeat-associated DSBs can efficiently lead to CAs and reshape the genome, they could be a rich source of evolutionary change.

Analysis of the genome and transcriptome of Cryptococcus neoformans var. grubii reveals complex RNA expression and microevolution leading to virulence attenuation.

Cryptococcus neoformans is a pathogenic basidiomycetous yeast responsible for more than 600,000 deaths each year. It occurs as two serotypes (A and D) representing two varieties (i.e. grubii and neoformans, respectively). Here, we sequenced the genome and performed an RNA-Seq-based analysis of the C. neoformans var. grubii transcriptome structure. We determined the chromosomal locations, analyzed the sequence/structural features of the centromeres, and identified origins of replication. The genome was annotated based on automated and manual curation. More than 40,000 introns populating more than 99% of the expressed genes were identified. Although most of these introns are located in the coding DNA sequences (CDS), over 2,000 introns in the untranslated regions (UTRs) were also identified. Poly(A)-containing reads were employed to locate the polyadenylation sites of more than 80% of the genes. Examination of the sequences around these sites revealed a new poly(A)-site-associated motif (AUGHAH). In addition, 1,197 miscRNAs were identified. These miscRNAs can be spliced and/or polyadenylated, but do not appear to have obvious coding capacities. Finally, this genome sequence enabled a comparative analysis of strain H99 variants obtained after laboratory passage. The spectrum of mutations identified provides insights into the genetics underlying the micro-evolution of a laboratory strain, and identifies mutations involved in stress responses, mating efficiency, and virulence.

The Pattern of Gene Amplification Is Determined by the Chromosomal Location of Hairpin-Capped Breaks

DNA palindromes often colocalize in cancer cells with chromosomal regions that are predisposed to gene amplification. The molecular mechanisms by which palindromes can cause gene amplification are largely unknown. Using yeast as a model system, we found that hairpin-capped double-strand breaks (DSBs) occurring at the location of human Alu-quasipalindromes lead to the formation of intrachromosomal amplicons with large inverted repeats (equivalent to homogeneously staining regions in mammalian chromosomes) or extrachromosomal palindromic molecules (equivalent to double minutes [DM] in mammalian cells). We demonstrate that the specific outcomes of gene amplification depend on the applied selection, the nature of the break, and the chromosomal location of the amplified gene relative to the site of the hairpin-capped DSB. The rules for the palindrome-dependent pathway of gene amplification defined in yeast may operate during the formation of amplicons in human tumors.

Gene expression divergence in yeast is coupled to evolution of DNA-encoded nucleosome organization

Eukaryotic transcription occurs within a chromatin environment, whose organization has an important regulatory function and is partly encoded in cis by the DNA sequence itself. Here, we examine whether evolutionary changes in gene expression are linked to changes in the DNA-encoded nucleosome organization of promoters. We find that in aerobic yeast species, where cellular respiration genes are active under typical growth conditions, the promoter sequences of these genes encode a relatively open (nucleosome-depleted) chromatin organization. This nucleosome-depleted organization requires only DNA sequence information, is independent of any cofactors and of transcription, and is a general property of growth-related genes. In contrast, in anaerobic yeast species, where cellular respiration genes are relatively inactive under typical growth conditions, respiration gene promoters encode relatively closed (nucleosome-occupied) chromatin organizations. Our results suggest a previously unidentified genetic mechanism underlying phenotypic diversity, consisting of DNA sequence changes that directly alter the DNA-encoded nucleosome organization of promoters.