PolyPhred: automating the detection and genotyping of single nucleotide substitutions using fluorescence-based resequencing.

Fluorescence-based sequencing is playing an increasingly important role in efforts to identify DNA polymorphisms and mutations of biological and medical interest. The application of this technology in generating the reference sequence of simple and complex genomes is also driving the development of new computer programs to automate base calling (Phred), sequence assembly (Phrap) and sequence assembly editing (Consed) in high throughput settings. In this report we describe a new computer program known as PolyPhred that automatically detects the presence of heterozygous single nucleotide substitutions by fluorescencebased sequencing of PCR products. Its operations are integrated with the use of the Phred, Phrap and Consed programs and together these tools generate a high throughput system for detecting DNA polymorphisms and mutations by large scale fluorescence-based resequencing. Analysis of sequences containing known DNA variants demonstrates that the accuracy of PolyPhred with single pass data is >99% when the sequences are generated with fluorescent dye-labeled primers and approximately 90% for those prepared with dye-labeled terminators.     

Evaluation of methods for de novo genome assembly from high-throughput sequencing reads reveals dependencies that affect the quality of the results

Recent developments in high-throughput sequencing technology have made low-cost sequencing an attractive approach for many genome analysis tasks. Increasing read lengths, improving quality and the production of increasingly larger numbers of usable sequences per instrument-run continue to make whole-genome assembly an appealing target application. In this paper we evaluate the feasibility of de novo genome assembly from short reads (≤100 nucleotides) through a detailed study involving genomic sequences of various lengths and origin, in conjunction with several of the currently popular assembly programs. Our extensive analysis demonstrates that, in addition to sequencing coverage, attributes such as the architecture of the target genome, the identity of the used assembly program, the average read length and the observed sequencing error rates are powerful variables that affect the best achievable assembly of the target sequence in terms of size and correctness.     

Assembly of overlapping DNA sequences by a program written in BASIC for 64K CP/M and MS-DOS IBM-compatible microcomputers.

The SEQALIGN programs1 described in this report aid in the assembly of up to 100 individual overlapping DNA sequences generated by M-13 subcloning and sequencing methods. The program produces a printout of the aligned sequences presented in register. Use of the program will be facilitated because 1) it is written with the Microsoft BASIC interpreter, 2) sequence data may be entered and edited using WORDSTAR or similar word processing programs, and 3) hardware requirements for execution of the program on CP/M or MS-DOS (IBM-PC compatible) systems are minimal.     

Shotgun sequencing,clone pooling,and comparative strategies for mapping and sequencing

Comparative genomic sequencing and analysis offers new wealth of information for target selection and the development of therapeutics. This article focuses on the following two key innovations in mapping and sequencing: first, shotgun sequencing of clone pools to combine the benefits of whole-genome shotgun and clone-by-clone strategies, and second, the leveraging of newly available assembled genomic sequences to improve the effectiveness of new sequencing projects through comparative mapping and comparative sequence assembly. The following specific sequencing and mapping methods are discussed in detail: clone-array pooled shotgun sequencing (CAPSS); transversal shotgun pooling designs; clone-array pooled shotgun mapping (CAPS-MAP); pooled genomic indexing (PGI); short-tag pooled genomic indexing (ST-PGI); and comparative sequence assembly (the CSA™ method). The methods can be implemented with only modest modifications of current large-scale sequencing pipelines and are highly synergistic with the next generation of sequencing technologies.     

Assembling millions of short DNA sequences using SSAKE

Novel DNA sequencing technologies with the potential for up to three orders magnitude more sequence throughput than conventional Sanger sequencing are emerging. The instrument now available from Solexa Ltd, produces millions of short DNA sequences of 25 nt each. Due to ubiquitous repeats in large genomes and the inability of short sequences to uniquely and unambiguously characterize them, the short read length limits applicability for de novo sequencing. However, given the sequencing depth and the throughput of this instrument, stringent assembly of highly identical sequences can be achieved. We describe SSAKE, a tool for aggressively assembling millions of short nucleotide sequences by progressively searching through a prefix tree for the longest possible overlap between any two sequences. SSAKE is designed to help leverage the information from short sequence reads by stringently assembling them into contiguous sequences that can be used to characterize novel sequencing targets. Availability: http://www.bcgsc.ca/bioinfo/software/ssake.     

SESAME (SEquence Sorter & AMplicon Explorer): genotyping based on high-throughput multiplex amplicon sequencing

SUMMARY: Characterizing genetic diversity through genotyping short amplicons is central to evolutionary biology. Next-generation sequencing (NGS) technologies changed the scale at which these type of data are acquired. SESAME is a web application package that assists genotyping of multiplexed individuals for several markers based on NGS amplicon sequencing. It automatically assigns reads to loci and individuals, corrects reads if standard samples are available and provides an intuitive graphical user interface (GUI) for allele validation based on the sequences and associated decision-making tools. The aim of SESAME is to help allele identification among a large number of sequences. AVAILABILITY: SESAME and its documentation are freely available under the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported Licence for Windows and Linux from http://www1.montpellier.inra.fr/CBGP/NGS/ or http://tinyurl.com/ngs-sesame.     

A novel DNAseq program for enhanced analysis of Illumina GAII data: a case study on antibody complementarity-determining regions

High-throughput DNA sequencing technologies are increasingly becoming powerful systems for the comprehensive analysis of variations in whole genomes or various DNA libraries. As they are capable of producing massive collections of short sequences with varying lengths, a major challenge is how to turn these reads into biologically meaningful information. The first stage is to assemble the short reads into longer sequences through an in silico process. However, currently available software/programs allow only the assembly of abundant sequences, which apparently results in the loss of highly variable (or rare) sequences or creates artefact assemblies. In this paper, we describe a novel program (DNAseq) that is capable of assembling highly variable sequences and displaying them directly for phylogenetic analysis. In addition, this program is Microsoft Windows-based and runs by a normal PC with 700MB RAM for a general use. We have applied it to analyse a human naive single-chain antibody (scFv) library, comprehensively revealing the diversity of antibody variable complementarity-determining regions (CDRs) and their families. Although only a scFv library was exemplified here, we envisage that this program could be applicable to other genome libraries.     

OSLay: optimal syntenic layout of unfinished assemblies

The whole genome shotgun approach to genome sequencing results in a collection of contigs that must be ordered and oriented to facilitate efficient gap closure. We present a new tool OSLay that uses synteny between matching sequences in a target assembly and a reference assembly to layout the contigs (or scaffolds) in the target assembly. The underlying algorithm is based on maximum weight matching. The tool provides an interactive visualization of the computed layout and the result can be imported into the assembly editing tool Consed to support the design of primer pairs for gap closure. MOTIVATION: To enhance efficiency in the gap closure phase of a genome project it is crucial to know which contigs are adjacent in the target genome. Related genome sequences can be used to layout contigs in an assembly. AVAILABILITY: OSLay is freely available from: http://www-ab.informatik.unituebingen.de/software/oslay.     

A strategy for assembling the maize (Zea mays L.) genome

Because the bulk of the maize (Zea mays L.) genome consists of repetitive sequences, sequencing efforts are being targeted to its 'gene-rich' fraction. Traditional assembly programs are inadequate for this approach because they are optimized for a uniform sampling of the genome and inherently lack the ability to differentiate highly similar paralogs. RESULTS: We report the development of bioinformatics tools for the accurate assembly of the maize genome. This software, which is based on innovative parallel algorithms to ensure scalability, assembled 730,974 genomic survey sequences fragments in 4 h using 64 Pentium III 1.26 GHz processors of a commodity cluster. Algorithmic innovations are used to reduce the number of pairwise alignments significantly without sacrificing quality. Clone pair information was used to estimate the error rate for improved differentiation of polymorphisms versus sequencing errors. The assembly was also used to evaluate the effectiveness of various filtering strategies and thereby provide information that can be used to focus subsequent sequencing efforts.     

Single-cell genomic sequencing using Multiple Displacement Amplification

Single microbial cells can now be sequenced using DNA amplified by the Multiple Displacement Amplification (MDA) reaction. The few femtograms of DNA in a bacterium are amplified into micrograms of high molecular weight DNA suitable for DNA library construction and Sanger sequencing. The MDA-generated DNA also performs well when used directly as template for pyrosequencing by the 454 Life Sciences method. While MDA from single cells loses some of the genomic sequence, this approach will greatly accelerate the pace of sequencing from uncultured microbes. The genetically linked sequences from single cells are also a powerful tool to be used in guiding genomic assembly of shotgun sequences of multiple organisms from environmental DNA extracts (metagenomic sequences).     

Gene-boosted assembly of a novel bacterial genome from very short reads

Recent improvements in technology have made DNA sequencing dramatically faster and more efficient than ever before. The new technologies produce highly accurate sequences, but one drawback is that the most efficient technology produces the shortest read lengths. Short-read sequencing has been applied successfully to resequence the human genome and those of other species but not to whole-genome sequencing of novel organisms. Here we describe the sequencing and assembly of a novel clinical isolate of Pseudomonas aeruginosa, strain PAb1, using very short read technology. From 8,627,900 reads, each 33 nucleotides in length, we assembled the genome into one scaffold of 76 ordered contiguous sequences containing 6,290,005 nucleotides, including one contig spanning 512,638 nucleotides, plus an additional 436 unordered contigs containing 416,897 nucleotides. Our method includes a novel gene-boosting algorithm that uses amino acid sequences from predicted proteins to build a better assembly. This study demonstrates the feasibility of very short read sequencing for the sequencing of bacterial genomes, particularly those for which a related species has been sequenced previously, and expands the potential application of this new technology to most known prokaryotic species.     

Whole-genome sequencing of eukaryotes: From sequencing of DNA fragments to a genome assembly

Rapid advances in sequencing technologies of second- and even third-generation made the whole genome sequencing a routine procedure. However, the methods for assembling of the obtained sequences and its results require special consideration. Modern assemblers are based on heuristic algorithms, which lead to fragmented genome assembly composed of scaffolds and contigs of different lengths, the order of which along the chromosome and belonging to a particular chromosome often remain unknown. In this regard, the resulting genome sequence can only be considered as a draft assembly. The principal improvement in the quality and reliability of a draft assembly can be achieved by targeted sequencing of the genome elements of different size, e.g., chromosomes, chromosomal regions, and DNA fragments cloned in different vectors, as well as using reference genome, optical mapping, and Hi-C technology. This approach, in addition to simplifying the assembly of the genome draft, will more accurately identify numerical and structural chromosomal variations and abnormalities of the genomes of the studied species. In this review, we discuss the key technologies for the genome sequencing and the de novo assembly, as well as different approaches to improve the quality of existing drafts of genome sequences.     

Probabilistic nucleotide assembling method for sequencing by hybridization

MOTIVATION: Developing a new method of assembling small sequences based on sequencing by hybridization with many positive and negative faults. First, an interpretation of a generic traveling salesman problem is provided (i.e. finding the shortest route for visiting many cities), using genetic algorithms. Second, positive errors are excluded before assembly by a sanitization process. RESULTS: The present method outperforms those described in previous studies, in terms of both time and accuracy. AVAILABILITY: http://kamit.med.u-tokai.ac.jp/~takaho/sbh/index.html     

Generation of Physical Map Contig-Specific Sequences Useful for Whole Genome Sequence Scaffolding

Along with the rapid advances of the nextgen sequencing technologies, more and more species are added to the list of organisms whose whole genomes are sequenced. However, the assembled draft genome of many organisms consists of numerous small contigs, due to the short length of the reads generated by nextgen sequencing platforms. In order to improve the assembly and bring the genome contigs together, more genome resources are needed. In this study, we developed a strategy to generate a valuable genome resource, physical map contig-specific sequences, which are randomly distributed genome sequences in each physical contig. Two-dimensional tagging method was used to create specific tags for 1,824 physical contigs, in which the cost was dramatically reduced. A total of 94,111,841 100-bp reads and 315,277 assembled contigs are identified containing physical map contig-specific tags. The physical map contig-specific sequences along with the currently available BAC end sequences were then used to anchor the catfish draft genome contigs. A total of 156,457 genome contigs (~79% of whole genome sequencing assembly) were anchored and grouped into 1,824 pools, in which 16,680 unique genes were annotated. The physical map contig-specific sequences are valuable resources to link physical map, genetic linkage map and draft whole genome sequences, consequently have the capability to improve the whole genome sequences assembly and scaffolding, and improve the genome-wide comparative analysis as well. The strategy developed in this study could also be adopted in other species whose whole genome assembly is still facing a challenge.     

Combination of de novo assembly of massive sequencing reads with classical repeat prediction improves identification of repetitive sequences in Schistosoma mansoni

The genome of the parasitic platyhelminth Schistosoma mansoni is composed of approximately 40% of repetitive sequences of which roughly 20% correspond to transposable elements. When the genome sequence became available, conventional repeat prediction programs were used to find these repeats, but only a fraction could be identified. To exhaustively characterize the repeats we applied a new massive sequencing based strategy: we re-sequenced the genome by next generation sequencing, aligned the sequencing reads to the genome and assembled all multiple-hit reads into contigs corresponding to the repetitive part of the genome. We present here, for the first time, this de novo repeat assembly strategy and we confirm that such assembly is feasible. We identified and annotated 4,143 new repeats in the S. mansoni genome. At least one third of the repeats are transcribed. This strategy allowed us also to identify 14 new microsatellite markers, which can be used for pedigree studies. Annotations and the combined (previously known and new) 5,420 repeat sequences (corresponding to 47% of the genome) are available for download (http://methdb.univ-perp.fr/downloads/).     

A sequence assembly and editing program for efficient management of large projects.

We describe a sequence assembly and editing program for managing large and small projects. It is being used to sequence complete cosmids and has substantially reduced the time taken to process the data. In addition to handling conventionally derived sequences it can use data obtained from Applied Biosystems,Inc. 373A and Pharmacia A.L.F. fluorescent sequencing machines. Readings are assembled automatically. All editing is performed using a mouse operated contig editor that displays aligned sequences and their traces together on the screen. The editor, which can be used on single contigs or for joining contigs, permits rapid movement along the aligned sequences. Insertions, deletions and replacements can be made in individual aligned readings and global changes can be made by editing the consensus. All changes are recorded. A click on a mouse button will display the traces covering the current cursor position, hence allowing quick resolution of problems. Another function automatically moves the cursor to the next unresolved character. The editor also provides facilities for annotating the sequences. Typical annotations include flagging the positions of primers used for walking, or for marking sites, such as compressions, that have caused problems during sequencing. Graphical displays aid the assessment of progress.     

Analysis of Medicago truncatula nodule expressed sequence tags

Systematic sequencing of expressed sequence tags (ESTs) can give a global picture of the assembly of genes involved in the development and function of organs. Indeterminate nodules representing different stages of the developmental program are especially suited to the study of organogenesis. With the vector lambdaHybriZAP, a cDNA library was constructed from emerging nodules of Medicago truncatula induced by Sinorhizobium meliloti. The 5' ends of 389 cDNA clones were sequenced, then these ESTs were analyzed both by sequence homology search and by studying their expression in roots and nodules. Two hundred fifty-six ESTs exhibited significant similarities to characterized data base entries and 40 of them represented 26 nodulin genes, while 133 had no similarity to sequences with known function. Only 60 out of the 389 cDNA clones corresponded to previously submitted M. truncatula EST sequences. For 117 cDNAs, reverse Northern (RNA) hybridization with root and nodule RNA probes revealed enhanced expression in the nodule, 48 clones are likely to code for novel nodulins, 33 cDNAs are clones of already known nodulin genes, and 36 clones exhibit similarity to other characterized genes. Thus, systematic analysis of the EST sequences and their expression patterns is a powerful way to identify nodule-specific and nodulation-related genes.     

Single molecule sequencing and genome assembly of a clinical specimen of Loa loa,the causative agent of loiasis

Background

More than 20% of the world’s population is at risk for infection by filarial nematodes and >180 million people worldwide are already infected. Along with infection comes significant morbidity that has a socioeconomic impact. The eight filarial nematodes that infect humans are Wuchereria bancrofti, Brugia malayi, Brugia timori, Onchocerca volvulus, Loa loa, Mansonella perstans, Mansonella streptocerca, and Mansonella ozzardi, of which three have published draft genome sequences. Since all have humans as the definitive host, standard avenues of research that rely on culturing and genetics have often not been possible. Therefore, genome sequencing provides an important window into understanding the biology of these parasites. The need for large amounts of high quality genomic DNA from homozygous, inbred lines; the availability of only short sequence reads from next-generation sequencing platforms at a reasonable expense; and the lack of random large insert libraries has limited our ability to generate high quality genome sequences for these parasites. However, the Pacific Biosciences single molecule, real-time sequencing platform holds great promise in reducing input amounts and generating sufficiently long sequences that bypass the need for large insert paired libraries.

Results

Here, we report on efforts to generate a more complete genome assembly for L. loa using genetically heterogeneous DNA isolated from a single clinical sample and sequenced on the Pacific Biosciences platform. To obtain the best assembly, numerous assemblers and sequencing datasets were analyzed, combined, and compared. Quiver-informed trimming of an assembly of only Pacific Biosciences reads by HGAP2 was selected as the final assembly of 96.4 Mbp in 2,250 contigs. This results in ~9% more of the genome in ~85% fewer contigs from ~80% less starting material at a fraction of the cost of previous Roche 454-based sequencing efforts.

Conclusions

The result is the most complete filarial nematode assembly produced thus far and demonstrates the utility of single molecule sequencing on the Pacific Biosciences platform for genetically heterogeneous metazoan genomes.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-788) contains supplementary material, which is available to authorized users.