Effective detection of remote homologues by searching in sequence dataset of a protein domain fold

Profile matching methods are commonly used in searches in protein sequence databases to detect evolutionary relationships. We describe here a sensitive protocol, which detects remote similarities by searching in a specialized database of sequences belonging to a fold. We have assessed this protocol by exploring the relationships we detect among sequences known to belong to specific folds. We find that searches within sequences adopting a fold are more effective in detecting remote similarities and evolutionary connections than searches in a database of all sequences. We also discuss the implications of using this strategy to link sequence and structure space.     

A protocol of automatic alignment of genome sequences using the program OWEN

A protocol of automatic hierarchical alignment of long DNA sequences using the program OWEN is described. The protocol is based on the command line regime of the program OWEN. The protocol makes it possible to align a large number of pairs of moderately similar sequences automatically. We used this protocol to align 8623 orthologous pairs of intergenic regions in human and murine genomes.     

Microarray and allele specific PCR detection of point mutations in Mycobacterium tuberculosis genes associated with drug resistance

Global public health is threatened by the emergence of potentially dangerous antibiotic drug-resistant strains of Mycobacterium tuberculosis. Point mutations in certain M. tuberculosis genes are associated with the resistance of M. tuberculosis strains to antibiotic drugs. The purpose of this study was to develop a suitable microarray-based protocol for the detection of point mutations in M. tuberculosis genes associated with drug resistance. We initially developed a conventional, oligonucleotide microarray protocol and used it to detect and identify on a single microarray slide a number of point mutation-containing rpoB and katG gene target sequences. However, the occurrence of some non-specific hybridization led us to the development of an improved protocol based on allele specific PCR combined with tags/anti-tags and microarrays. This protocol was evaluated by detecting point mutations in M. tuberculosis katG and rpoB gene templates produced by recombinant PCR. The methodology allowed sequences containing single point mutations to be readily distinguished from wild type sequences. The data obtained with the improved protocol had strong and specific signals and relatively low amounts of non-specific hybridization. We successfully used this protocol to detect and identify (<8 h) a number of clinically relevant point mutations in the rpoB, katG and rpsL genes of M. tuberculosis clinical isolates. Our allele specific PCR/tags and anti-tags/microarray protocol has several advantages over our conventional oligonucleotide microarray protocol, and it may have broad applications for point mutation detection.     

Enrichment of meiotic recombination hotspot sequences by avidin capture technology

About 40% of the hotspots for meiotic recombination contain the degenerate consensus sequence 5′-CCNCCNTNNCCNC-3′. Here we present a novel protocol for enriching hotspot sequences from digested genomic DNA by using biotinylated oligonucleotides and streptavidin-coated magnetic beads. The captured hotspots can be released by simple digestion with restriction enzymes for subsequent characterization by second generation sequencing or PCR. The capture protocol specifically enriches hotspot sequences, judged by using fluorophore-conjugated synthetic oligonucleotides and synthetic double-stranded oligonucleotides in combination with PCR. The capture protocol enriches single-stranded DNA, denatured double-stranded DNA, and large fragments (> 3000 bp) of digested plasmid DNA with good efficacy. No false positive and false negatives were detected when enriching digested DNA from human cell cultures and primary human cells. The protocol can probably be adapted to enriching sequences other than the hotspot sequence by altering the sequence in the capture oligonucleotide. We intend to apply this protocol in studies assessing effects of micronutrient status on meiotic recombination events in human sperm.     

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly effective at mapping highly repetitive sequences to heterochromatic regions, where computational approaches face prohibitive challenges. Here we describe a streamlined protocol for DNA ISH that circumvents formamide washes that are standard steps in other DNA ISH protocols. Our protocol is optimized for hybridization with short single strand DNA probes that carry fluorescent dyes, which effectively mark repetitive DNA sequences within heterochromatic chromosomal regions across a number of different insect tissue types. However, applications may be extended to use with larger probes and visualization of single copy (non-repetitive) DNA sequences. We demonstrate this method by mapping several different repetitive sequences to squashed chromosomes from Drosophila melanogaster neural cells and Nasonia vitripennis spermatocytes. We show hybridization patterns for both small, commercially synthesized probes and for a larger probe for comparison. This procedure uses simple laboratory supplies and reagents, and is ideal for investigators who have little experience with performing DNA ISH.     

Mark–recapture cloning: a straightforward and cost‐effective cloning method for population genetics of single‐copy nuclear DNA sequences in diploids

We describe a simple protocol to reduce the number of cloning reactions of nuclear DNA sequences in population genetic studies of diploid organisms. Cloning is a necessary step to obtain correct haplotypes in such organisms, and, while traditional methods are efficient at cloning together many genes of a single individual, population geneticists rather need to clone the same locus in many individuals. Our method consists of marking individual sequences during the polymerase chain reaction (PCR) using 5′‐tailed primers with small polynucleotide tags. PCR products are mixed together before the cloning reaction and clones are sequenced with universal plasmid primers. The individual from which a sequence comes from is identified by the tag sequences upstream of each initial primer. We called our protocol mark–recapture (MR) cloning. We present results from 57 experiments of MR cloning conducted in four distinct laboratories using nuclear loci of various lengths in different invertebrate species. Rate of capture (proportion of individuals for which one or more sequences were retrieved) and multiple capture (proportion of individuals for which two or more sequences were retrieved) empirically obtained are described. We estimated that MR cloning allowed reducing costs by up to 70% when compared to conventional individual‐based cloning. However, we recommend to adjust the mark:recapture ratio in order to obtain multiple sequences from the same individual and circumvent inherent technical artefacts of PCR, cloning and sequencing. We argue that MR cloning is a valid and reliable high‐throughput method, providing the number of sequences exceeds the number of individuals initially amplified.     

Optimisation of a Microsatellite Enrichment Technique in Saccharum spp.

The creation of enriched microsatellite libraries can ensure an abundant supply of microsatellite sequences at a considerably reduced cost. We report here the modification to an existing enrichment protocol and the optimisation of this technique for use on specific plant species, in this case, sugarcane. Using this optimised protocol, we achieved a 40% increase in the level of enrichment.     

Efficient and high-throughput vector construction and Agrobacterium-mediated transformation of Arabidopsis thaliana suspension-cultured cells for functional genomics

We established a large-scale, high-throughput protocol to construct Arabidopsis thaliana suspension-cultured cell lines, each of which carries a single transgene, using Agrobacterium-mediated transformation. We took advantage of RIKEN Arabidopsis full-length (RAFL) cDNA clones and the Gateway cloning system for high-throughput preparation of binary vectors carrying individual full-length cDNA sequences. Throughout all cloning steps, multiple-well plates were used to treat 96 samples simultaneously in a high-throughput manner. The optimal conditions for Agrobacterium-mediated transformation of 96 independent binary vector constructs were established to obtain transgenic cell lines efficiently. We evaluated the protocol by generating transgenic Arabidopsis T87 cell lines carrying individual 96 metabolism-related RAFL cDNA fragments, and showed that the protocol was useful for high-throughput and large-scale production of gain-of-function lines for functional genomics.     

Designing and optimizing comparative anchor primers for comparative gene mapping and phylogenetic inference

Here we describe protocols for designing, optimizing and implementing conserved anchor primers for use in genome mapping or phylogenetic applications, with particular emphasis on homologous gene sequences among mammals. The increasing number of whole genome sequences in public databases makes this approach applicable across a wide range of taxa. Genome sequences from representatives of two or more divergent subclades within a taxonomic group of interest are used to identify candidate local alignments (i.e., exons, exons spanning introns or conserved 5'- or 3'-untranslated regions) that contain sequences with appropriate variability for the chosen downstream application. PCR primers are designed to maximize amplification success across a broad range of taxa, and are optimized under a touchdown thermocycling protocol. Based on the initial optimization results, primers are selected for application in a diverse sampling of species, or for mapping the genome of a target species of interest. We discuss factors that have to be considered for experimental design of broad-scope phylogenetic studies. With this protocol, primers can be designed, optimized and implemented within as little as 1-2 weeks.     

Feasibility in the inverse protein folding protocol.

Methods for protein structure (3D)-sequence (1D) compatibility evaluation (threading) have been developed during the past decade. The protocol in which a sequence can recognize its compatible structure in the structural library (i.e., the fold recognition or the forward-folding search) is available for the structure prediction of new proteins. However, the reverse protocol, in which a structure recognizes its homologous sequences among a sequence database, named the inverse-folding search, is a more difficult application. In this study, we have investigated the feasibility of the latter approach. A structural library, composed of about 400 well-resolved structures with mutually dissimilar sequences, was prepared, and 163 of them had remote homologs in the library. We examined whether they could correctly seek their homologs by both forward- and inverse-folding searches. The results showed that the inverse-folding protocol is more effective than the forward-folding protocol, once the reference states of the compatibility functions are appropriately adjusted. This adjustment only slightly affects the ability of the forward-folding search. We noticed that the scoring, in which a given sequence is re-mounted onto a structure according to the 3D-1D alignment determined by the dynamic programming method, is only effective in the forward-folding protocol and not in the inverse-folding protocol. Namely, the inverse-folding search works significantly better with the score given by the 3D-1D alignment per se, rather than that obtained by the re-mounting. The implications of these results are discussed.     

Fluorescent in situ hybridisation to amphioxus chromosomes

We describe an efficient protocol for mapping genes and other DNA sequences to amphioxus chromosomes using fluorescent in situ hybridisation. We apply this method to identify the number and location of ribosomal DNA gene clusters and telomere sequences in metaphase spreads of Branchiostoma floridae. We also describe how the locations of two single copy genes can be mapped relative to each other, and demonstrate this by mapping an amphioxus Pax gene relative to a homologue of the Notch gene. These methods have great potential for performing comparative genomics between amphioxus and vertebrates.     

AmpliconDuo: A Split-Sample Filtering Protocol for High-Throughput Amplicon Sequencing of Microbial Communities

High throughput sequencing (HTSeq) of small ribosomal subunit amplicons has the potential for a comprehensive characterization of microbial community compositions, down to rare species. However, the error-prone nature of the multi-step experimental process requires that the resulting raw sequences are subjected to quality control procedures. These procedures often involve an abundance cutoff for rare sequences or clustering of sequences, both of which limit genetic resolution. Here we propose a simple experimental protocol that retains the high genetic resolution granted by HTSeq methods while effectively removing many low abundance sequences that are likely due to PCR and sequencing errors. According to this protocol, we split samples and submit both halves to independent PCR and sequencing runs. The resulting sequence data is graphically and quantitatively characterized by the discordance between the two experimental branches, allowing for a quick identification of problematic samples. Further, we discard sequences that are not found in both branches (“AmpliconDuo filter”). We show that the majority of sequences removed in this way, mostly low abundance but also some higher abundance sequences, show features expected from random modifications of true sequences as introduced by PCR and sequencing errors. On the other hand, the filter retains many low abundance sequences observed in both branches and thus provides a more reliable census of the rare biosphere. We find that the AmpliconDuo filter increases biological resolution as it increases apparent community similarity between biologically similar communities, while it does not affect apparent community similarities between biologically dissimilar communities. The filter does not distort overall apparent community compositions. Finally, we quantitatively explain the effect of the AmpliconDuo filter by a simple mathematical model.     

Use of DNA-protein interaction to isolate specific genomic DNA sequences.

We describe a simple and rapid method for the isolation of specific genomic DNA sequences recognized by DNA-binding proteins. This procedure consists of four steps: (1) restriction enzyme digestion and size fractionation of genomic DNA; (2) DNA--protein binding using the gel mobility-shift assay; (3) ligation of isolated DNA fragments followed by transformation of Escherichia coli; and (4) screening of recombinant clones for inserts containing specific DNA--protein binding sequences. We have used this protocol to isolate human DNA sequences, 100-200 bp in size, that are recognized by both partially purified and affinity purified proteins. Unlike other procedures designed to identify genomic target sequences, the method described does not require polymerase chain reaction or successive immunoprecipitations.     

Computational protein design and large-scale assessment by I-TASSER structure assembly simulations

Protein design aims at designing new protein molecules of desired structure and functionality. One of the major obstacles to large-scale protein design are the extensive time and manpower requirements for experimental validation of designed sequences. Recent advances in protein structure prediction have provided potentials for an automated assessment of the designed sequences via folding simulations. We present a new protocol for protein design and validation. The sequence space is initially searched by Monte Carlo sampling guided by a public atomic potential, with candidate sequences selected by the clustering of sequence decoys. The designed sequences are then assessed by I-TASSER folding simulations, which generate full-length atomic structural models by the iterative assembly of threading fragments. The protocol is tested on 52 nonhomologous single-domain proteins, with an average sequence identity of 24% between the designed sequences and the native sequences. Despite this low sequence identity, three-dimensional models predicted for the first designed sequence have an RMSD of < 2 Å to the target structure in 62% of cases. This percentage increases to 77% if we consider the three-dimensional models from the top 10 designed sequences. Such a striking consistency between the target structure and the structural prediction from nonhomologous sequences, despite the fact that the design and folding algorithms adopt completely different force fields, indicates that the design algorithm captures the features essential to the global fold of the target. On average, the designed sequences have a free energy that is 0.39 kcal/(mol residue) lower than in the native sequences, potentially affording a greater stability to synthesized target folds.     

A universal protocol to generate consensus level genome sequences for foot-and-mouth disease virus and other positive-sense polyadenylated RNA viruses using the Illumina MiSeq

Background

Next-Generation Sequencing (NGS) is revolutionizing molecular epidemiology by providing new approaches to undertake whole genome sequencing (WGS) in diagnostic settings for a variety of human and veterinary pathogens. Previous sequencing protocols have been subject to biases such as those encountered during PCR amplification and cell culture, or are restricted by the need for large quantities of starting material. We describe here a simple and robust methodology for the generation of whole genome sequences on the Illumina MiSeq. This protocol is specific for foot-and-mouth disease virus (FMDV) or other polyadenylated RNA viruses and circumvents both the use of PCR and the requirement for large amounts of initial template.

Results

The protocol was successfully validated using five FMDV positive clinical samples from the 2001 epidemic in the United Kingdom, as well as a panel of representative viruses from all seven serotypes. In addition, this protocol was successfully used to recover 94% of an FMDV genome that had previously been identified as cell culture negative. Genome sequences from three other non-FMDV polyadenylated RNA viruses (EMCV, ERAV, VESV) were also obtained with minor protocol amendments. We calculated that a minimum coverage depth of 22 reads was required to produce an accurate consensus sequence for FMDV O. This was achieved in 5 FMDV/O/UKG isolates and the type O FMDV from the serotype panel with the exception of the 5′ genomic termini and area immediately flanking the poly(C) region.

Conclusions

We have developed a universal WGS method for FMDV and other polyadenylated RNA viruses. This method works successfully from a limited quantity of starting material and eliminates the requirement for genome-specific PCR amplification. This protocol has the potential to generate consensus-level sequences within a routine high-throughput diagnostic environment.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-828) contains supplementary material, which is available to authorized users.     

Two-dimensional gel analysis of complex DNA families: Methodology and apparatus

We describe a reproducible protocol for the analysis of individual members of complex mammalian gene families by gel fractionation in two dimensions within a specially designed, easily built electrophoretic apparatus. We have used this protocol to resolve the family of mouseH-2 class I genes, with approximately 30 members, as well as two different families of endogenous retroviral-like sequences, each of which has approximately 180 members dispersed throughout the genome. The results demonstrate the feasibility of using this protocol for rapid, whole genome analysis of individual animals and cell lines. Two-dimensional DNA analysis of highly repeated retroviral-like DNA families could be applied to genetic mapping and cloning experiments as well as to obtaining whole genome fingerprints in the analysis of somatic cell hybrid lines that contain a subset of chromosomes from the genome of interest.