Big domains are novel Ca²+-binding modules: evidences from big domains of Leptospira immunoglobulin-like (Lig) proteins

Background

Many bacterial surface exposed proteins mediate the host-pathogen interaction more effectively in the presence of Ca²⁺. Leptospiral immunoglobulin-like (Lig) proteins, LigA and LigB, are surface exposed proteins containing Bacterial immunoglobulin like (Big) domains. The function of proteins which contain Big fold is not known. Based on the possible similarities of immunoglobulin and βγ-crystallin folds, we here explore the important question whether Ca²⁺ binds to a Big domains, which would provide a novel functional role of the proteins containing Big fold.

Principal Findings

We selected six individual Big domains for this study (three from the conserved part of LigA and LigB, denoted as Lig A3, Lig A4, and LigBCon5; two from the variable region of LigA, i.e., 9^th (Lig A9) and 10^th repeats (Lig A10); and one from the variable region of LigB, i.e., LigBCen2. We have also studied the conserved region covering the three and six repeats (LigBCon1-3 and LigCon). All these proteins bind the calcium-mimic dye Stains-all. All the selected four domains bind Ca²⁺ with dissociation constants of 2–4 µM. Lig A9 and Lig A10 domains fold well with moderate thermal stability, have β-sheet conformation and form homodimers. Fluorescence spectra of Big domains show a specific doublet (at 317 and 330 nm), probably due to Trp interaction with a Phe residue. Equilibrium unfolding of selected Big domains is similar and follows a two-state model, suggesting the similarity in their fold.

Conclusions

We demonstrate that the Lig are Ca²⁺-binding proteins, with Big domains harbouring the binding motif. We conclude that despite differences in sequence, a Big motif binds Ca²⁺. This work thus sets up a strong possibility for classifying the proteins containing Big domains as a novel family of Ca²⁺-binding proteins. Since Big domain is a part of many proteins in bacterial kingdom, we suggest a possible function these proteins via Ca²⁺ binding.     

Unlocking Short Read Sequencing for Metagenomics

Background

Different high-throughput nucleic acid sequencing platforms are currently available but a trade-off currently exists between the cost and number of reads that can be generated versus the read length that can be achieved.

Methodology/Principal Findings

We describe an experimental and computational pipeline yielding millions of reads that can exceed 200 bp with quality scores approaching that of traditional Sanger sequencing. The method combines an automatable gel-less library construction step with paired-end sequencing on a short-read instrument. With appropriately sized library inserts, mate-pair sequences can overlap, and we describe the SHERA software package that joins them to form a longer composite read.

Conclusions/Significance

This strategy is broadly applicable to sequencing applications that benefit from low-cost high-throughput sequencing, but require longer read lengths. We demonstrate that our approach enables metagenomic analyses using the Illumina Genome Analyzer, with low error rates, and at a fraction of the cost of pyrosequencing.     

Impacts of low coverage depths and post-mortem DNA damage on variant calling: a simulation study

Background

Massively parallel sequencing platforms, featuring high throughput and relatively short read lengths, are well suited to ancient DNA (aDNA) studies. Variant identification from short-read alignment could be hindered, however, by low DNA concentrations common to historic samples, which constrain sequencing depths, and post-mortem DNA damage patterns.

Results

We simulated pairs of sequences to act as reference and sample genomes at varied GC contents and divergence levels. Short-read sequence pools were generated from sample sequences, and subjected to varying levels of “post-mortem” damage by adjusting levels of fragmentation and fragmentation biases, transition rates at sequence ends, and sequencing depths. Mapping of sample read pools to reference sequences revealed several trends, including decreased alignment success with increased read length and decreased variant recovery with increased divergence. Variants were generally called with high accuracy, however identification of SNPs (single-nucleotide polymorphisms) was less accurate for high damage/low divergence samples. Modest increases in sequencing depth resulted in rapid gains in total variant recovery, and limited improvements to recovery of heterozygous variants.

Conclusions

This in silico study suggests aDNA-associated damage patterns minimally impact variant call accuracy and recovery from short-read alignment, while modest increases in sequencing depth can greatly improve variant recovery.

     

Pebble and Rock Band: Heuristic Resolution of Repeats and Scaffolding in the Velvet Short-Read de Novo Assembler

Background

Despite the short length of their reads, micro-read sequencing technologies have shown their usefulness for de novo sequencing. However, especially in eukaryotic genomes, complex repeat patterns are an obstacle to large assemblies.

Principal Findings

We present a novel heuristic algorithm, Pebble, which uses paired-end read information to resolve repeats and scaffold contigs to produce large-scale assemblies. In simulations, we can achieve weighted median scaffold lengths (N50) of above 1 Mbp in Bacteria and above 100 kbp in more complex organisms. Using real datasets we obtained a 96 kbp N50 in Pseudomonas syringae and a unique 147 kbp scaffold of a ferret BAC clone. We also present an efficient algorithm called Rock Band for the resolution of repeats in the case of mixed length assemblies, where different sequencing platforms are combined to obtain a cost-effective assembly.

Conclusions

These algorithms extend the utility of short read only assemblies into large complex genomes. They have been implemented and made available within the open-source Velvet short-read de novo assembler.     

Mapping the landscape of tandem repeat variability by targeted long read single molecule sequencing in familial X-linked intellectual disability

Background

The etiology of more than half of all patients with X-linked intellectual disability remains elusive, despite array-based comparative genomic hybridization, whole exome or genome sequencing. Since short read massive parallel sequencing approaches do not allow the detection of larger tandem repeat expansions, we hypothesized that such expansions could be a hidden cause of X-linked intellectual disability.

Methods

We selectively captured over 1800 tandem repeats on the X chromosome and characterized them by long read single molecule sequencing in 3 families with idiopathic X-linked intellectual disability.

Results

In male DNA samples, full tandem repeat length sequences were obtained for 88–93% of the targets and up to 99.6% of the repeats with a moderate guanine-cytosine content. Read length and analysis pipeline allow to detect cases of >?900?bp tandem repeat expansion. In one family, one repeat expansion co-occurs with down-regulation of the neighboring MIR222 gene. This gene has previously been implicated in intellectual disability and is apparently linked to FMR1 and NEFH overexpression associated with neurological disorders.

Conclusions

This study demonstrates the power of single molecule sequencing to measure tandem repeat lengths and detect expansions, and suggests that tandem repeat mutations may be a hidden cause of X-linked intellectual disability.

     

Hobbes: optimized gram-based methods for efficient read alignment

Recent advances in sequencing technology have enabled the rapid generation of billions of bases at relatively low cost. A crucial first step in many sequencing applications is to map those reads to a reference genome. However, when the reference genome is large, finding accurate mappings poses a significant computational challenge due to the sheer amount of reads, and because many reads map to the reference sequence approximately but not exactly. We introduce Hobbes, a new gram-based program for aligning short reads, supporting Hamming and edit distance. Hobbes implements two novel techniques, which yield substantial performance improvements: an optimized gram-selection procedure for reads, and a cache-efficient filter for pruning candidate mappings. We systematically tested the performance of Hobbes on both real and simulated data with read lengths varying from 35 to 100 bp, and compared its performance with several state-of-the-art read-mapping programs, including Bowtie, BWA, mrsFast and RazerS. Hobbes is faster than all other read mapping programs we have tested while maintaining high mapping quality. Hobbes is about five times faster than Bowtie and about 2–10 times faster than BWA, depending on read length and error rate, when asked to find all mapping locations of a read in the human genome within a given Hamming or edit distance, respectively. Hobbes supports the SAM output format and is publicly available at http://hobbes.ics.uci.edu.     

Multiplexed highly-accurate DNA sequencing of closely-related HIV-1 variants using continuous long reads from single molecule,real-time sequencing

Single Molecule, Real-Time (SMRT^®) Sequencing (Pacific Biosciences, Menlo Park, CA, USA) provides the longest continuous DNA sequencing reads currently available. However, the relatively high error rate in the raw read data requires novel analysis methods to deconvolute sequences derived from complex samples. Here, we present a workflow of novel computer algorithms able to reconstruct viral variant genomes present in mixtures with an accuracy of >QV50. This approach relies exclusively on Continuous Long Reads (CLR), which are the raw reads generated during SMRT Sequencing. We successfully implement this workflow for simultaneous sequencing of mixtures containing up to forty different >9 kb HIV-1 full genomes. This was achieved using a single SMRT Cell for each mixture and desktop computing power. This novel approach opens the possibility of solving complex sequencing tasks that currently lack a solution.     

Simple tools for assembling and searching high-density picolitre pyrophosphate sequence data

Background

The advent of pyrophosphate sequencing makes large volumes of sequencing data available at a lower cost than previously possible. However, the short read lengths are difficult to assemble and the large dataset is difficult to handle. During the sequencing of a virus from the tsetse fly, Glossina pallidipes, we found the need for tools to search quickly a set of reads for near exact text matches.

Methods

A set of tools is provided to search a large data set of pyrophosphate sequence reads under a "live" CD version of Linux on a standard PC that can be used by anyone without prior knowledge of Linux and without having to install a Linux setup on the computer. The tools permit short lengths of de novo assembly, checking of existing assembled sequences, selection and display of reads from the data set and gathering counts of sequences in the reads.

Results

Demonstrations are given of the use of the tools to help with checking an assembly against the fragment data set; investigating homopolymer lengths, repeat regions and polymorphisms; and resolving inserted bases caused by incomplete chain extension.

Conclusion

The additional information contained in a pyrophosphate sequencing data set beyond a basic assembly is difficult to access due to a lack of tools. The set of simple tools presented here would allow anyone with basic computer skills and a standard PC to access this information.     

Modeling the Cost-Effectiveness of the Integrated Disease Surveillance and Response (IDSR) System: Meningitis in Burkina Faso

Background

Effective surveillance for infectious diseases is an essential component of public health. There are few studies estimating the cost-effectiveness of starting or improving disease surveillance. We present a cost-effectiveness analysis the Integrated Disease Surveillance and Response (IDSR) strategy in Africa.

Methodology/Principal Findings

To assess the impact of the IDSR in Africa, we used pre- and post- IDSR meningococcal meningitis surveillance data from Burkina Faso (1996–2002 and 2003–2007). IDSR implementation was correlated with a median reduction of 2 weeks to peak of outbreaks (25^th percentile 1 week; 75^th percentile 4 weeks). IDSR was also correlated with a reduction of 43 meningitis cases per 100,000 (25^th–40: 75^th-129). Assuming the correlations between reductions in time to peak of outbreaks and cases are related, the cost-effectiveness of IDSR was $23 per case averted (25^th-$30; 75^th - cost saving), and $98 per meningitis-related death averted (25^th-$140: 75^th – cost saving).

Conclusions/Significance

We cannot absolutely claim that the measured differences were due to IDSR. We believe, however, that it is reasonable to claim that IDSR can improve the cost-effectiveness of public health surveillance.     

A Bead-based Normalization for Uniform Sequencing depth (BeNUS) protocol for multi-samples sequencing exemplified by HLA-B

Background

Human leukocyte antigen (HLA) is a group of genes that are extremely polymorphic among individuals and populations and have been associated with more than 100 different diseases and adverse drug effects. HLA typing is accordingly an important tool in clinical application, medical research, and population genetics. We have previously developed a phase-defined HLA gene sequencing method using MiSeq sequencing.

Results

Here we report a simple, high-throughput, and cost-effective sequencing method that includes normalized library preparation and adjustment of DNA molar concentration. We applied long-range PCR to amplify HLA-B for 96 samples followed by transposase-based library construction and multiplex sequencing with the MiSeq sequencer. After sequencing, we observed low variation in read percentages (0.2% to 1.55%) among the 96 demultiplexed samples. On this basis, all the samples were amenable to haplotype phasing using our phase-defined sequencing method. In our study, a sequencing depth of 800x was necessary and sufficient to achieve full phasing of HLA-B alleles with reliable assignment of the allelic sequence to the 8 digit level.

Conclusions

Our HLA sequencing method optimized for 96 multiplexing samples is highly time effective and cost effective and is especially suitable for automated multi-sample library preparation and sequencing.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-645) contains supplementary material, which is available to authorized users.     

AT1 Receptor Induced Alterations in Histone H2A Reveal Novel Insights into GPCR Control of Chromatin Remodeling

Chronic activation of angiotensin II (AngII) type 1 receptor (AT₁R), a prototypical G protein-coupled receptor (GPCR) induces gene regulatory stress which is responsible for phenotypic modulation of target cells. The AT₁R-selective drugs reverse the gene regulatory stress in various cardiovascular diseases. However, the molecular mechanisms are not clear. We speculate that activation states of AT₁R modify the composition of histone isoforms and post-translational modifications (PTM), thereby alter the structure-function dynamics of chromatin. We combined total histone isolation, FPLC separation, and mass spectrometry techniques to analyze histone H2A in HEK293 cells with and without AT₁R activation. We have identified eight isoforms: H2AA, H2AG, H2AM, H2AO, H2AQ, {"type":"entrez-protein","attrs":{"text":"Q96QV6","term_id":"74752099","term_text":"Q96QV6"}}Q96QV6, H2AC and H2AL. The isoforms, H2AA, H2AC and H2AQ were methylated and H2AC was phosphorylated. The relative abundance of specific H2A isoforms and PTMs were further analyzed in relationship to the activation states of AT₁R by immunochemical studies. Within 2 hr, the isoforms, H2AA/O exchanged with H2AM. The monomethylated H2AC increased rapidly and the phosphorylated H2AC decreased, thus suggesting that enhanced H2AC methylation is coupled to Ser1p dephosphorylation. We show that H2A125Kme1 promotes interaction with the heterochromatin associated protein, HP1α. These specific changes in H2A are reversed by treatment with the AT₁R specific inhibitor losartan. Our analysis provides a first step towards an awareness of histone code regulation by GPCRs.     

SVachra: a tool to identify genomic structural variation in mate pair sequencing data containing inward and outward facing reads

Background

Characterization of genomic structural variation (SV) is essential to expanding the research and clinical applications of genome sequencing. Reliance upon short DNA fragment paired end sequencing has yielded a wealth of single nucleotide variants and internal sequencing read insertions-deletions, at the cost of limited SV detection. Multi-kilobase DNA fragment mate pair sequencing has supplemented the void in SV detection, but introduced new analytic challenges requiring SV detection tools specifically designed for mate pair sequencing data. Here, we introduce SVachra – Structural Variation Assessment of CHRomosomal Aberrations, a breakpoint calling program that identifies large insertions-deletions, inversions, inter- and intra-chromosomal translocations utilizing both inward and outward facing read types generated by mate pair sequencing.

Results

We demonstrate SVachra’s utility by executing the program on large-insert (Illumina Nextera) mate pair sequencing data from the personal genome of a single subject (HS1011). An additional data set of long-read (Pacific BioSciences RSII) was also generated to validate SV calls from SVachra and other comparison SV calling programs. SVachra exhibited the highest validation rate and reported the widest distribution of SV types and size ranges when compared to other SV callers.

Conclusions

SVachra is a highly specific breakpoint calling program that exhibits a more unbiased SV detection methodology than other callers.

     

Read Length and Repeat Resolution: Exploring Prokaryote Genomes Using Next-Generation Sequencing Technologies

Background

There are a growing number of next-generation sequencing technologies. At present, the most cost-effective options also produce the shortest reads. However, even for prokaryotes, there is uncertainty concerning the utility of these technologies for the de novo assembly of complete genomes. This reflects an expectation that short reads will be unable to resolve small, but presumably abundant, repeats.

Methodology/Principal Findings

Using a simple model of repeat assembly, we develop and test a technique that, for any read length, can estimate the occurrence of unresolvable repeats in a genome, and thus predict the number of gaps that would need to be closed to produce a complete sequence. We apply this technique to 818 prokaryote genome sequences. This provides a quantitative assessment of the relative performance of various lengths. Notably, unpaired reads of only 150nt can reconstruct approximately 50% of the analysed genomes with fewer than 96 repeat-induced gaps. Nonetheless, there is considerable variation amongst prokaryotes. Some genomes can be assembled to near contiguity using very short reads while others require much longer reads.

Conclusions

Given the diversity of prokaryote genomes, a sequencing strategy should be tailored to the organism under study. Our results will provide researchers with a practical resource to guide the selection of the appropriate read length.     

Systematic Monitoring of Male Circumcision Scale-Up in Nyanza,Kenya: Exploratory Factor Analysis of Service Quality Instrument and Performance Ranking

Background

Considerable conceptual and operational complexities related to service quality measurements and variability in delivery contexts of scaled-up medical male circumcision, pose real challenges to monitoring implementation of quality and safety. Clarifying latent factors of the quality instruments can enhance contextual applicability and the likelihood that observed service outcomes are appropriately assessed.

Objective

To explore factors underlying SYMMACS service quality assessment tool (adopted from the WHO VMMC quality toolkit) and; determine service quality performance using composite quality index derived from the latent factors.

Study design

Using a comparative process evaluation of Voluntary Medical Male Circumcision Scale-Up in Kenya site level data was collected among health facilities providing VMMC over two years. Systematic Monitoring of the Medical Male Circumcision Scale-Up quality instrument was used to assess availability of guidelines, supplies and equipment, infection control, and continuity of care services. Exploratory factor analysis was performed to clarify quality structure.

Results

Fifty four items and 246 responses were analyzed. Based on Eigenvalue >1.00 cut-off, factors 1, 2 & 3 were retained each respectively having eigenvalues of 5.78; 4.29; 2.99. These cumulatively accounted for 29.1% of the total variance (12.9%; 9.5%; 6.7%) with final communality estimates being 13.06. Using a cut-off factor loading value of ≥0.4, fifteen items loading on factor 1, five on factor 2 and one on factor 3 were retained. Factor 1closely relates to preparedness to deliver safe male circumcisions while factor two depicts skilled task performance and compliance with protocols. Of the 28 facilities, 32% attained between 90^th and 95^th percentile (excellent); 45% between 50^th and 75^th percentiles (average) and 14.3% below 25^th percentile (poor).

Conclusion

the service quality assessment instrument may be simplified to have nearly 20 items that relate more closely to service outcomes. Ranking of facilities and circumcision procedure using a composite index based on these items indicates that majority performed above average.     

Methane production and sulfate reduction in two Appalachian peatlands

Anaerobic carbon mineralization was evaluated over a 1-year period in two Sphagnum-dominated peatlands, Big Run Bog, West Virginia, and Buckle's Bog, Maryland. In the top 35 cm of peat, mean rates of methane production, anaerobic carbon dioxide production, and sulfate reduction at Big Run Bog were 63,406 and 146 mol L^-1 d^-1, respectively, and at Buckle's Bog were 18, 486 and 104 mol L^-1 d^-1. Annual anaerobic carbon mineralization to methane and carbon dioxide at Big Run Bog and Buckle's Bog was 52.8 and 57.2 mol m^-2, respectively. Rates of methane production were similar to rates reported for other freshwater peatlands, but methane production accounted for only 11.7 and 2.8%, respectively, of the total anaerobic carbon mineralization at these two sites. Carbon dioxide production, resulting substantially from sulfate reduction, dominated anaerobic carbon mineralization. Considerable sulfate reduction despite low instantaneous dissolved sulfate concentrations (typically < 300 mol L^-1 of substrate) was apparently fueled by oxidation and rapid turnover of the reduced inorganic sulfur pool.The coincidence of high sulfate inputs to the Big Run Bog and Buckle's Bog watersheds through acid precipitation with the unexpected importance of sulfate reduction leads us to suggest a new hypothesis: peatlands not receiving high sulfate loading should exhibit low rates of anaerobic decomposition, and a predominance of methane production over sulfate reduction; however, if such peatlands become subjected to high rates of sulfur deposition, sulfate reduction may be enhanced as an anaerobic mineralization pathway with attendant effects on carbon balance and peat accumulation.