采用诱导环化酶制备Illumina Mate-paired DNA文库

新一代测序技术(NGS)的文库制备方法在基因组的拼装中起着重要作用。但是NGS技术制备的普通DNA文库片段只有500 bp左右,难以满足复杂基因组的从头(de novo)拼装要求。三代测序技术的读长可以达到20 kb,但是其高错误率及测序成本过高使得其又不易推广。因此二代测序的Mate-paired文库制备技术一直在基因组的de novo拼装中扮演着非常重要的角色。目前主流的NGS平台Illumina制备的Mate-paired文库的片段范围只有2~5 kb,为了得到更长的可用于Illumina平台测序的Mate-paired文库,本研究首次整合并优化了Illumina和Roche/454两种测序平台的Mate-paired文库制备技术,采用诱导环化酶来提高基因组长片段DNA的环化效率,成功建立了20 kb Mate-paired文库制备技术,并已将该技术应用于人类基因组20 kb Mate-paired文库制备。该技术为Illumina平台制备长片段Mate-paired库提供了方法指导。     

Is the whole greater than the sum of its parts? De novo assembly strategies for bacterial genomes based on paired-end sequencing

Background

Whole genome sequence construction is becoming increasingly feasible because of advances in next generation sequencing (NGS), including increasing throughput and read length. By simply overlapping paired-end reads, we can obtain longer reads with higher accuracy, which can facilitate the assembly process. However, the influences of different library sizes and assembly methods on paired-end sequencing-based de novo assembly remain poorly understood.

Results

We used 250 bp Illumina Miseq paired-end reads of different library sizes generated from genomic DNA from Escherichia coli DH1 and Streptococcus parasanguinis FW213 to compare the assembly results of different library sizes and assembly approaches. Our data indicate that overlapping paired-end reads can increase read accuracy but sometimes cause insertion or deletions. Regarding genome assembly, merged reads only outcompete original paired-end reads when coverage depth is low, and larger libraries tend to yield better assembly results. These results imply that distance information is the most critical factor during assembly. Our results also indicate that when depth is sufficiently high, assembly from subsets can sometimes produce better results.

Conclusions

In summary, this study provides systematic evaluations of de novo assembly from paired end sequencing data. Among the assembly strategies, we find that overlapping paired-end reads is not always beneficial for bacteria genome assembly and should be avoided or used with caution especially for genomes containing high fraction of repetitive sequences. Because increasing numbers of projects aim at bacteria genome sequencing, our study provides valuable suggestions for the field of genomic sequence construction.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1859-8) contains supplementary material, which is available to authorized users.     

Sequencing,annotation and comparative analysis of nine BACs of the giant panda(Ailuropoda melanoleuca)

A 10-fold BAC library for the giant panda was constructed and nine BACs were selected to generate finish sequences.These BACs could be used as a validation resource for the de novo assembly accuracy of the whole genome shotgun sequencing reads of the giant panda newly generated by Illumina GA sequencing technology.Complete Sanger sequencing,assembly,annotation and comparative analysis were carried out on the selected BACs of a joint length 878 kb.Homologue search and de novo prediction methods were used to ...     

Whole-genome sequencing and assembly with high-throughput, short-read technologies

While recently developed short-read sequencing technologies may dramatically reduce the sequencing cost and eventually achieve the $1000 goal for re-sequencing, their limitations prevent the de novo sequencing of eukaryotic genomes with the standard shotgun sequencing protocol. We present SHRAP (SHort Read Assembly Protocol), a sequencing protocol and assembly methodology that utilizes high-throughput short-read technologies. We describe a variation on hierarchical sequencing with two crucial differences: (1) we select a clone library from the genome randomly rather than as a tiling path and (2) we sample clones from the genome at high coverage and reads from the clones at low coverage. We assume that 200 bp read lengths with a 1% error rate and inexpensive random fragment cloning on whole mammalian genomes is feasible. Our assembly methodology is based on first ordering the clones and subsequently performing read assembly in three stages: (1) local assemblies of regions significantly smaller than a clone size, (2) clone-sized assemblies of the results of stage 1, and (3) chromosome-sized assemblies. By aggressively localizing the assembly problem during the first stage, our method succeeds in assembling short, unpaired reads sampled from repetitive genomes. We tested our assembler using simulated reads from D. melanogaster and human chromosomes 1, 11, and 21, and produced assemblies with large sets of contiguous sequence and a misassembly rate comparable to other draft assemblies. Tested on D. melanogaster and the entire human genome, our clone-ordering method produces accurate maps, thereby localizing fragment assembly and enabling the parallelization of the subsequent steps of our pipeline. Thus, we have demonstrated that truly inexpensive de novo sequencing of mammalian genomes will soon be possible with high-throughput, short-read technologies using our methodology.     

SMRT sequencing only de novo assembly of the sugar beet (Beta vulgaris) chloroplast genome

Background

Third generation sequencing methods, like SMRT (Single Molecule, Real-Time) sequencing developed by Pacific Biosciences, offer much longer read length in comparison to Next Generation Sequencing (NGS) methods. Hence, they are well suited for de novo- or re-sequencing projects. Sequences generated for these purposes will not only contain reads originating from the nuclear genome, but also a significant amount of reads originating from the organelles of the target organism. These reads are usually discarded but they can also be used for an assembly of organellar replicons. The long read length supports resolution of repetitive regions and repeats within the organelles genome which might be problematic when just using short read data. Additionally, SMRT sequencing is less influenced by GC rich areas and by long stretches of the same base.

Results

We describe a workflow for a de novo assembly of the sugar beet (Beta vulgaris ssp. vulgaris) chloroplast genome sequence only based on data originating from a SMRT sequencing dataset targeted on its nuclear genome. We show that the data obtained from such an experiment are sufficient to create a high quality assembly with a higher reliability than assemblies derived from e.g. Illumina reads only. The chloroplast genome is especially challenging for de novo assembling as it contains two large inverted repeat (IR) regions. We also describe some limitations that still apply even though long reads are used for the assembly.

Conclusions

SMRT sequencing reads extracted from a dataset created for nuclear genome (re)sequencing can be used to obtain a high quality de novo assembly of the chloroplast of the sequenced organism. Even with a relatively small overall coverage for the nuclear genome it is possible to collect more than enough reads to generate a high quality assembly that outperforms short read based assemblies. However, even with long reads it is not always possible to clarify the order of elements of a chloroplast genome sequence reliantly which we could demonstrate with Fosmid End Sequences (FES) generated with Sanger technology. Nevertheless, this limitation also applies to short read sequencing data but is reached in this case at a much earlier stage during finishing.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-015-0726-6) contains supplementary material, which is available to authorized users.     

Restriction site associated DNA (RAD) for de novo sequencing and marker discovery in sugarcane borer,Diatraea saccharalis Fab. (Lepidoptera: Crambidae)

We present the development of a genomic library using RADseq (restriction site associated DNA sequencing) protocol for marker discovery that can be applied on evolutionary studies of the sugarcane borer Diatraea saccharalis, an important South American insect pest. A RADtag protocol combined with Illumina paired‐end sequencing allowed de novo discovery of 12 811 SNPs and a high‐quality assembly of 122.8M paired‐end reads from six individuals, representing 40 Gb of sequencing data. Approximately 1.7 Mb of the sugarcane borer genome distributed over 5289 minicontigs were obtained upon assembly of second reads from first reads RADtag loci where at least one SNP was discovered and genotyped. Minicontig lengths ranged from 200 to 611 bp and were used for functional annotation and microsatellite discovery. These markers will be used in future studies to understand gene flow and adaptation to host plants and control tactics.     

Pseudo-Sanger sequencing: massively parallel production of long and near error-free reads using NGS technology

Background

Usually, next generation sequencing (NGS) technology has the property of ultra-high throughput but the read length is remarkably short compared to conventional Sanger sequencing. Paired-end NGS could computationally extend the read length but with a lot of practical inconvenience because of the inherent gaps. Now that Illumina paired-end sequencing has the ability of read both ends from 600 bp or even 800 bp DNA fragments, how to fill in the gaps between paired ends to produce accurate long reads is intriguing but challenging.

Results

We have developed a new technology, referred to as pseudo-Sanger (PS) sequencing. It tries to fill in the gaps between paired ends and could generate near error-free sequences equivalent to the conventional Sanger reads in length but with the high throughput of the Next Generation Sequencing. The major novelty of PS method lies on that the gap filling is based on local assembly of paired-end reads which have overlaps with at either end. Thus, we are able to fill in the gaps in repetitive genomic region correctly. The PS sequencing starts with short reads from NGS platforms, using a series of paired-end libraries of stepwise decreasing insert sizes. A computational method is introduced to transform these special paired-end reads into long and near error-free PS sequences, which correspond in length to those with the largest insert sizes. The PS construction has 3 advantages over untransformed reads: gap filling, error correction and heterozygote tolerance. Among the many applications of the PS construction is de novo genome assembly, which we tested in this study. Assembly of PS reads from a non-isogenic strain of Drosophila melanogaster yields an N50 contig of 190 kb, a 5 fold improvement over the existing de novo assembly methods and a 3 fold advantage over the assembly of long reads from 454 sequencing.

Conclusions

Our method generated near error-free long reads from NGS paired-end sequencing. We demonstrated that de novo assembly could benefit a lot from these Sanger-like reads. Besides, the characteristic of the long reads could be applied to such applications as structural variations detection and metagenomics.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-14-711) contains supplementary material, which is available to authorized users.     

ConDeTri--a content dependent read trimmer for Illumina data

During the last few years, DNA and RNA sequencing have started to play an increasingly important role in biological and medical applications, especially due to the greater amount of sequencing data yielded from the new sequencing machines and the enormous decrease in sequencing costs. Particularly, Illumina/Solexa sequencing has had an increasing impact on gathering data from model and non-model organisms. However, accurate and easy to use tools for quality filtering have not yet been established. We present ConDeTri, a method for content dependent read trimming for next generation sequencing data using quality scores of each individual base. The main focus of the method is to remove sequencing errors from reads so that sequencing reads can be standardized. Another aspect of the method is to incorporate read trimming in next-generation sequencing data processing and analysis pipelines. It can process single-end and paired-end sequence data of arbitrary length and it is independent from sequencing coverage and user interaction. ConDeTri is able to trim and remove reads with low quality scores to save computational time and memory usage during de novo assemblies. Low coverage or large genome sequencing projects will especially gain from trimming reads. The method can easily be incorporated into preprocessing and analysis pipelines for Illumina data. AVAILABILITY AND IMPLEMENTATION: Freely available on the web at http://code.google.com/p/condetri.     

A chromosome‐scale reference genome and genome‐wide genetic variations elucidate adaptation in yak

Yak is an important livestock animal for the people indigenous to the harsh, oxygen‐limited Qinghai‐Tibetan Plateau and Hindu Kush ranges of the Himalayas. The yak genome was sequenced in 2012, but its assembly was fragmented because of the inherent limitations of the Illumina sequencing technology used to analyse it. An accurate and complete reference genome is essential for the study of genetic variations in this species. Long‐read sequences are more complete than their short‐read counterparts and have been successfully applied towards high‐quality genome assembly for various species. In this study, we present a high‐quality chromosome‐scale yak genome assembly (BosGru_PB_v1.0) constructed with long‐read sequencing and chromatin interaction technologies. Compared to an existing yak genome assembly (BosGru_v2.0), BosGru_PB_v1.0 shows substantially improved chromosome sequence continuity, reduced repetitive structure ambiguity, and gene model completeness. To characterize genetic variation in yak, we generated de novo genome assemblies based on Illumina short reads for seven recognized domestic yak breeds in Tibet and Sichuan and one wild yak from Hoh Xil. We compared these eight assemblies to the BosGru_PB_v1.0 genome, obtained a comprehensive map of yak genetic diversity at the whole‐genome level, and identified several protein‐coding genes absent from the BosGru_PB_v1.0 assembly. Despite the genetic bottleneck experienced by wild yak, their diversity was nonetheless higher than that of domestic yak. Here, we identified breed‐specific sequences and genes by whole‐genome alignment, which may facilitate yak breed identification.     

Identification of Optimum Sequencing Depth Especially for De Novo Genome Assembly of Small Genomes Using Next Generation Sequencing Data

Next Generation Sequencing (NGS) is a disruptive technology that has found widespread acceptance in the life sciences research community. The high throughput and low cost of sequencing has encouraged researchers to undertake ambitious genomic projects, especially in de novo genome sequencing. Currently, NGS systems generate sequence data as short reads and de novo genome assembly using these short reads is computationally very intensive. Due to lower cost of sequencing and higher throughput, NGS systems now provide the ability to sequence genomes at high depth. However, currently no report is available highlighting the impact of high sequence depth on genome assembly using real data sets and multiple assembly algorithms. Recently, some studies have evaluated the impact of sequence coverage, error rate and average read length on genome assembly using multiple assembly algorithms, however, these evaluations were performed using simulated datasets. One limitation of using simulated datasets is that variables such as error rates, read length and coverage which are known to impact genome assembly are carefully controlled. Hence, this study was undertaken to identify the minimum depth of sequencing required for de novo assembly for different sized genomes using graph based assembly algorithms and real datasets. Illumina reads for E.coli (4.6 MB) S.kudriavzevii (11.18 MB) and C.elegans (100 MB) were assembled using SOAPdenovo, Velvet, ABySS, Meraculous and IDBA-UD. Our analysis shows that 50X is the optimum read depth for assembling these genomes using all assemblers except Meraculous which requires 100X read depth. Moreover, our analysis shows that de novo assembly from 50X read data requires only 6–40 GB RAM depending on the genome size and assembly algorithm used. We believe that this information can be extremely valuable for researchers in designing experiments and multiplexing which will enable optimum utilization of sequencing as well as analysis resources.     

基于短序列测序数据的四倍体拟南芥转录组研究

为了促进对四倍体拟南芥(A.suecica)的研究,阐明多倍体植物在染色体加倍过程中遗传物质的变化,从而在分子层面上解释多倍体植物的环境适应和进化机制,描述了一套基于第二代测序技术的转录组短序列组装和生物信息学分析方法.通过对23 000 000条来至于Illumina测序平台的序列数据进行SOAPdenovo组装,以...     

Paired-End Sequencing of Long-Range DNA Fragments for De Novo Assembly of Large,Complex Mammalian Genomes by Direct Intra-Molecule Ligation

Background

The relatively short read lengths from next generation sequencing (NGS) technologies still pose a challenge for de novo assembly of complex mammal genomes. One important solution is to use paired-end (PE) sequence information experimentally obtained from long-range DNA fragments (>1 kb). Here, we characterize and extend a long-range PE library construction method based on direct intra-molecule ligation (or molecular linker-free circularization) for NGS.

Results

We found that the method performs stably for PE sequencing of 2- to 5- kb DNA fragments, and can be extended to 10–20 kb (and even in extremes, up to ∼35 kb). We also characterized the impact of low quality input DNA on the method, and develop a whole-genome amplification (WGA) based protocol using limited input DNA (<1 µg). Using this PE dataset, we accurately assembled the YanHuang (YH) genome, the first sequenced Asian genome, into a scaffold N50 size of >2 Mb, which is over100-times greater than the initial size produced with only small insert PE reads(17 kb). In addition, we mapped two 7- to 8- kb insertions in the YH genome using the larger insert sizes of the long-range PE data.

Conclusions

In conclusion, we demonstrate here the effectiveness of this long-range PE sequencing method and its use for the de novo assembly of a large, complex genome using NGS short reads.     

Complete genome sequence of Corynebacterium pseudotuberculosis I19, a strain isolated from a cow in Israel with bovine mastitis

This work reports the completion and annotation of the genome sequence of Corynebacterium pseudotuberculosis I19, isolated from an Israeli dairy cow with severe clinical mastitis. To present the whole-genome sequence, a de novo assembly approach using 33 million short (25-bp) mate-paired SOLiD reads only was applied. Furthermore, the automatic, functional, and manual annotations were attained with the use of several algorithms in a multistep process.     

Generation of long insert pairs using a Cre-LoxP Inverse PCR approach

Large insert mate pair reads have a major impact on the overall success of de novo assembly and the discovery of inherited and acquired structural variants. The positional information of mate pair reads generally improves genome assembly by resolving repeat elements and/or ordering contigs. Currently available methods for building such libraries have one or more of limitations, such as relatively small insert size; unable to distinguish the junction of two ends; and/or low throughput. We developed a new approach, Cre-LoxP Inverse PCR Paired-End (CLIP-PE), which exploits the advantages of (1) Cre-LoxP recombination system to efficiently circularize large DNA fragments, (2) inverse PCR to enrich for the desired products that contain both ends of the large DNA fragments, and (3) the use of restriction enzymes to introduce a recognizable junction site between ligated fragment ends and to improve the self-ligation efficiency. We have successfully created CLIP-PE libraries up to 22 kb that are rich in informative read pairs and low in small fragment background. These libraries have demonstrated the ability to improve genome assemblies. The CLIP-PE methodology can be implemented with existing and future next-generation sequencing platforms.     

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.     

Chromosome‐level hybrid de novo genome assemblies as an attainable option for nonmodel insects

The emergence of third‐generation sequencing (3GS; long‐reads) is bringing closer the goal of chromosome‐size fragments in de novo genome assemblies. This allows the exploration of new and broader questions on genome evolution for a number of nonmodel organisms. However, long‐read technologies result in higher sequencing error rates and therefore impose an elevated cost of sufficient coverage to achieve high enough quality. In this context, hybrid assemblies, combining short‐reads and long‐reads, provide an alternative efficient and cost‐effective approach to generate de novo, chromosome‐level genome assemblies. The array of available software programs for hybrid genome assembly, sequence correction and manipulation are constantly being expanded and improved. This makes it difficult for nonexperts to find efficient, fast and tractable computational solutions for genome assembly, especially in the case of nonmodel organisms lacking a reference genome or one from a closely related species. In this study, we review and test the most recent pipelines for hybrid assemblies, comparing the model organism Drosophila melanogaster to a nonmodel cactophilic Drosophila, D. mojavensis. We show that it is possible to achieve excellent contiguity on this nonmodel organism using the dbg2olc pipeline.     

A complete genome sequence for Pseudomonas syringae pv. pisi PP1 highlights the importance of multiple modes of horizontal gene transfer during phytopathogen evolution

Hybrid assembly strategies that combine long-read sequencing reads from Oxford Nanopore's MinION device combined with high-depth Illumina paired-end reads have enabled completion and circularization of both plasmids and chromosomes from multiple bacterial strains. Here we demonstrate the utility of supplementing Illumina paired-end reads from a previously published draft genome of P. syringae pv. pisi PP1 with long reads to generate a complete genome sequence for this strain. The phylogenetic placement and genomic repertoire of virulence factors within this strain provides a unique perspective on virulence evolution within P. syringae phylogroup 2, and highlights that strains can rapidly acquire virulence factors through horizontal gene transfer by acquisition of plasmids as well as through chromosomal recombination.