基于PC/Linux的核酸序列分析系统的构建及其应用

基于PC机和Linux操作系统, 利用Phred/Phrap/Consed软件和Blast软件, 构建了核酸序列大规模自动分析系统. 该套系统可自动完成从测序峰图向核酸序列的转化、载体序列去除、序列自动拼接、重复序列鉴定以及序列的相似性分析, 可加速对大规模测序数据的分析和利用.     

Lineage-Specific Biology Revealed by a Finished Genome Assembly of the Mouse

The mouse (Mus musculus) is the premier animal model for understanding human disease and development. Here we show that a comprehensive understanding of mouse biology is only possible with the availability of a finished, high-quality genome assembly. The finished clone-based assembly of the mouse strain C57BL/6J reported here has over 175,000 fewer gaps and over 139 Mb more of novel sequence, compared with the earlier MGSCv3 draft genome assembly. In a comprehensive analysis of this revised genome sequence, we are now able to define 20,210 protein-coding genes, over a thousand more than predicted in the human genome (19,042 genes). In addition, we identified 439 long, non–protein-coding RNAs with evidence for transcribed orthologs in human. We analyzed the complex and repetitive landscape of 267 Mb of sequence that was missing or misassembled in the previously published assembly, and we provide insights into the reasons for its resistance to sequencing and assembly by whole-genome shotgun approaches. Duplicated regions within newly assembled sequence tend to be of more recent ancestry than duplicates in the published draft, correcting our initial understanding of recent evolution on the mouse lineage. These duplicates appear to be largely composed of sequence regions containing transposable elements and duplicated protein-coding genes; of these, some may be fixed in the mouse population, but at least 40% of segmentally duplicated sequences are copy number variable even among laboratory mouse strains. Mouse lineage-specific regions contain 3,767 genes drawn mainly from rapidly-changing gene families associated with reproductive functions. The finished mouse genome assembly, therefore, greatly improves our understanding of rodent-specific biology and allows the delineation of ancestral biological functions that are shared with human from derived functions that are not.     

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.     

A comparative analysis of HGSC and Celera human genome assemblies and gene sets

MOTIVATION: Since the simultaneous publication of the human genome assembly by the International Human Genome Sequencing Consortium (HGSC) and Celera Genomics, several comparisons have been made of various aspects of these two assemblies. In this work, we set out to provide a more comprehensive comparative analysis of the two assemblies and their associated gene sets. RESULTS: The local sequence content for both draft genome assemblies has been similar since the early releases, however it took a year for the quality of the Celera assembly to approach that of HGSC, suggesting an advantage of HGSC's hierarchical shotgun (HS) sequencing strategy over Celera's whole genome shotgun (WGS) approach. While similar numbers of ab initio predicted genes can be derived from both assemblies, Celera's Otto approach consistently generated larger, more varied gene sets than the Ensembl gene build system. The presence of a non-overlapping gene set has persisted with successive data releases from both groups. Since most of the unique genes from either genome assembly could be mapped back to the other assembly, we conclude that the gene set discrepancies do not reflect differences in local sequence content but rather in the assemblies and especially the different gene-prediction methodologies.     

Revision of the Capsaspora genome using read mating information adjusts the view on premetazoan genome

The genome sequences of unicellular holozoans, the closest relatives to animals, are shedding light on the evolution of animal multicellularity, shaping the genetic contents of the putative premetazoans. However, the assembly quality of the genomes remains poor compared to the major model organisms such as human and fly. Improving the assembly is critical for precise comparative genomics studies and further molecular biological studies requiring accurate sequence information such as enhancer analysis and genome editing. In this report, we present a new strategy to improve the assembly by fully exploiting the information of Illumina mate-pair reads. By visualizing the distance and orientation of the mapped read pairs, we could highlight the regions where possible assembly errors exist in the genome sequence of Capsaspora, a lineage of unicellular holozoans. Manual modification of these errors repaired 590 assembly problems in total and reassembled 84 supercontigs into 55. Our telomere prediction analysis using the read pairs containing the pan-eukaryotic telomere-like sequence identified at least 13 chromosomes. The resulting new assembly posed us a re-annotation of 112 genes, including 15 putative receptor protein tyrosine kinases. Our strategy thus provides a useful approach for improving assemblies of draft genomes, and the new Capsaspora genome offers us an opportunity to adjust the view on the genome of the unicellular animal ancestor.     

A high-resolution radiation hybrid map of rhesus macaque chromosome 5 identifies rearrangements in the genome assembly

A 10,000-rad radiation hybrid (RH) cell panel of the rhesus macaque was generated to construct a comprehensive RH map of chromosome 5. The map represents 218 markers typed in 185 RH clones. The 4846-cR map has an average marker spacing of 798 kb. Alignments of the RH map to macaque and human genome sequences confirm a large inversion and reveal a previously unreported telomeric inversion. The macaque genome sequence indicates small translocations from the ancestral homolog of macaque chromosome 5 to macaque chromosomes 1 and 6. The RH map suggests that these are probably assembly artifacts. Unlike the genome sequence, the RH mapping data indicate the conservation of synteny between macaque chromosome 5 and human chromosome 4. This study shows that the 10,000-rad panel is appropriate for the generation of a high-resolution whole-genome RH map suitable for the verification of the rhesus genome assembly.     

Recent segmental and gene duplications in the mouse genome

Background

The high quality of the mouse genome draft sequence and its associated annotations are an invaluable biological resource. Identifying recent duplications in the mouse genome, especially in regions containing genes, may highlight important events in recent murine evolution. In addition, detecting recent sequence duplications can reveal potentially problematic regions of the genome assembly. We use BLAST-based computational heuristics to identify large (≥ 5 kb) and recent (≥ 90% sequence identity) segmental duplications in the mouse genome sequence. Here we present a database of recently duplicated regions of the mouse genome found in the mouse genome sequencing consortium (MGSC) February 2002 and February 2003 assemblies.

Results

We determined that 33.6 Mb of 2,695 Mb (1.2%) of sequence from the February 2003 mouse genome sequence assembly is involved in recent segmental duplications, which is less than that observed in the human genome (around 3.5-5%). From this dataset, 8.9 Mb (26%) of the duplication content consisted of 'unmapped' chromosome sequence. Moreover, we suspect that an additional 18.5 Mb of sequence is involved in duplication artifacts arising from sequence misassignment errors in this genome assembly. By searching for genes that are located within these regions, we identified 675 genes that mapped to duplicated regions of the mouse genome. Sixteen of these genes appear to have been duplicated independently in the human genome. From our dataset we further characterized a 42 kb recent segmental duplication of Mater, a maternal-effect gene essential for embryogenesis in mice.

Conclusion

Our results provide an initial analysis of the recently duplicated sequence and gene content of the mouse genome. Many of these duplicated loci, as well as regions identified to be involved in potential sequence misassignment errors, will require further mapping and sequencing to achieve accuracy. A Genome Browser database was set up to display the identified duplication content presented in this work. This data will also be relevant to the growing number of investigators who use the draft genome sequence for experimental design and analysis.

     

Management and visualization of whole genome shotgun assemblies using SAM

We have designed and implemented a system to manage whole genome shotgun sequences and whole genome sequence assembly data flow. The Sequence Assembly Manager (SAM) consists primarily of a MySQL relational database and Perl applications designed to easily manipulate and coordinate the analysis of sequence information and to view and report genome assembly progress through its Common Gateway Interface (CGI) web interface. The application includes a tool to compare sequence assemblies to fingerprint maps that has been used successfully to improve and validate both maps and sequence assemblies of the Rhodococcus sp.RHAI and Cryptococcus neoformans WM276 genomes.     

Succinct data structures for assembling large genomes

MOTIVATION: Second-generation sequencing technology makes it feasible for many researches to obtain enough sequence reads to attempt the de novo assembly of higher eukaryotes (including mammals). De novo assembly not only provides a tool for understanding wide scale biological variation, but within human biomedicine, it offers a direct way of observing both large-scale structural variation and fine-scale sequence variation. Unfortunately, improvements in the computational feasibility for de novo assembly have not matched the improvements in the gathering of sequence data. This is for two reasons: the inherent computational complexity of the problem and the in-practice memory requirements of tools. RESULTS: In this article, we use entropy compressed or succinct data structures to create a practical representation of the de Bruijn assembly graph, which requires at least a factor of 10 less storage than the kinds of structures used by deployed methods. Moreover, because our representation is entropy compressed, in the presence of sequencing errors it has better scaling behaviour asymptotically than conventional approaches. We present results of a proof-of-concept assembly of a human genome performed on a modest commodity server.     

Shotgun haplotyping: a novel method for surveying allelic sequence variation

Haplotypic sequences contain significantly more information than genotypes of genetic markers and are critical for studying disease association and genome evolution. Current methods for obtaining haplotypic sequences require the physical separation of alleles before sequencing, are time consuming and are not scaleable for large surveys of genetic variation. We have developed a novel method for acquiring haplotypic sequences from long PCR products using simple, high-throughput techniques. This method applies modified shotgun sequencing protocols to sequence both alleles concurrently, with read-pair information allowing the two alleles to be separated during sequence assembly. Although the haplotypic sequences can be assembled manually from the resultant data using pre-existing sequence assembly software, we have devised a novel heuristic algorithm to automate assembly and remove human error. We validated the approach on two long PCR products amplified from the human genome and confirmed the accuracy of our sequences against full-length clones of the same alleles. This method presents a simple high-throughput means to obtain full haplotypic sequences potentially up to 20 kb in length and is suitable for surveying genetic variation even in poorly-characterized genomes as it requires no prior information on sequence variation.     

Assessment of the relationship between signal intensities and transcript concentration for Affymetrix GeneChip®arrays

Background

The availability of both mouse and human draft genomes has marked the beginning of a new era of comparative mammalian genomics. The two available mouse genome assemblies, from the public mouse genome sequencing consortium and Celera Genomics, were obtained using different clone libraries and different assembly methods.

Results

We present here a critical comparison of the two latest mouse genome assemblies. The utility of the combined genomes is further demonstrated by comparing them with the human 'golden path' and through a subsequent analysis of a resulting conserved sequence element (CSE) database, which allows us to identify over 6,000 potential novel genes and to derive independent estimates of the number of human protein-coding genes.

Conclusion

The Celera and public mouse assemblies differ in about 10% of the mouse genome. Each assembly has advantages over the other: Celera has higher accuracy in base-pairs and overall higher coverage of the genome; the public assembly, however, has higher sequence quality in some newly finished bacterial artifical chromosome clone (BAC) regions and the data are freely accessible. Perhaps most important, by combining both assemblies, we can get a better annotation of the human genome; in particular, we can obtain the most complete set of CSEs, one third of which are related to known genes and some others are related to other functional genomic regions. More than half the CSEs are of unknown function. From the CSEs, we estimate the total number of human protein-coding genes to be about 40,000. This searchable publicly available online CSEdb will expedite new discoveries through comparative genomics.     

Review of general algorithmic features for genome assemblers for next generation sequencers

In the realm of bioinformatics and computational biology,the most rudimentary data upon which all the analysis is built is the sequence data of genes,proteins and RNA.The sequence data of the entire genome is the solution to the genome assembly problem.The scope of this contribution is to provide an overview on the art of problem-solving applied within the domain of genome assembly in the nextgeneration sequencing(NGS) platforms.This article discusses the major genome assemblers that were proposed in the literature during the past decade by outlining their basic working principles.It is intended to act as a qualitative,not a quantitative,tutorial to all working on genome assemblers pertaining to the next generation of sequencers.We discuss the theoretical aspects of various genome assemblers,identifying their working schemes.We also discuss briefly the direction in which the area is headed towards along with discussing core issues on software simplicity.     

Update of the <Emphasis Type="Italic">Anopheles gambiae</Emphasis>PEST genome assembly

Background  

The genome of Anopheles gambiae, the major vector of malaria, was sequenced and assembled in 2002. This initial genome assembly and analysis made available to the scientific community was complicated by the presence of assembly issues, such as scaffolds with no chromosomal location, no sequence data for the Y chromosome, haplotype polymorphisms resulting in two different genome assemblies in limited regions and contaminating bacterial DNA.     

The diploid genome sequence of an individual human

Presented here is a genome sequence of an individual human. It was produced from ∼32 million random DNA fragments, sequenced by Sanger dideoxy technology and assembled into 4,528 scaffolds, comprising 2,810 million bases (Mb) of contiguous sequence with approximately 7.5-fold coverage for any given region. We developed a modified version of the Celera assembler to facilitate the identification and comparison of alternate alleles within this individual diploid genome. Comparison of this genome and the National Center for Biotechnology Information human reference assembly revealed more than 4.1 million DNA variants, encompassing 12.3 Mb. These variants (of which 1,288,319 were novel) included 3,213,401 single nucleotide polymorphisms (SNPs), 53,823 block substitutions (2–206 bp), 292,102 heterozygous insertion/deletion events (indels)(1–571 bp), 559,473 homozygous indels (1–82,711 bp), 90 inversions, as well as numerous segmental duplications and copy number variation regions. Non-SNP DNA variation accounts for 22% of all events identified in the donor, however they involve 74% of all variant bases. This suggests an important role for non-SNP genetic alterations in defining the diploid genome structure. Moreover, 44% of genes were heterozygous for one or more variants. Using a novel haplotype assembly strategy, we were able to span 1.5 Gb of genome sequence in segments >200 kb, providing further precision to the diploid nature of the genome. These data depict a definitive molecular portrait of a diploid human genome that provides a starting point for future genome comparisons and enables an era of individualized genomic information.     

NSIT: Novel Sequence Identification Tool

Novel sequences are DNA sequences present in an individual''s genome but absent in the human reference assembly. They are predicted to be biologically important, both individual and population specific, and consistent with the known human migration paths. Recent works have shown that an average person harbors 2–5 Mb of such sequences and estimated that the human pan-genome contains as high as 19–40 Mb of novel sequences. To identify them in a de novo genome assembly, some existing sequence aligners have been used but no computational method has been specifically proposed for this task. In this work, we developed NSIT (Novel Sequence Identification Tool), a software that can accurately and efficiently identify novel sequences in an individual''s de novo whole genome assembly. We identified and characterized 1.1 Mb, 1.2 Mb, and 1.0 Mb of novel sequences in NA18507 (African), YH (Asian), and NA12878 (European) de novo genome assemblies, respectively. Our results show very high concordance with the previous work using the respective reference assembly. In addition, our results using the latest human reference assembly suggest that the amount of novel sequences per individual may not be as high as previously reported. We additionally developed a graphical viewer for comparisons of novel sequence contents. The viewer also helped in identifying sequence contamination; we found 130 kb of Epstein-Barr virus sequence in the previously published NA18507 novel sequences as well as 287 kb of zebrafish repeats in NA12878 de novo assembly. NSIT requires 2GB of RAM and 1.5–2 hrs on a commodity desktop. The program is applicable to input assemblies with varying contig/scaffold sizes, ranging from 100 bp to as high as 50 Mb. It works in both 32-bit and 64-bit systems and outperforms, by large margins, other fast sequence aligners previously applied to this task. To our knowledge, NSIT is the first software designed specifically for novel sequence identification in a de novo human genome assembly.     

Establishment of an eHAP1 human haploid cell line hybrid reference genome assembled from short and long reads

Haploid cell lines are a valuable research tool with broad applicability for genetic assays. As such the fully haploid human cell line, eHAP1, has been used in a wide array of studies. However, the absence of a corresponding reference genome sequence for this cell line has limited the potential for more widespread applications to experiments dependent on available sequence, like capture-clone methodologies. We generated ~15× coverage Nanopore long reads from ten GridION flowcells and utilized this data to assemble a de novo draft genome using minimap and miniasm and subsequently polished using Racon. This assembly was further polished using previously generated, low-coverage, Illumina short reads with Pilon and ntEdit. This resulted in a hybrid eHAP1 assembly with >90% complete BUSCO scores. We further assessed the eHAP1 long read data for structural variants using Sniffles and identify a variety of rearrangements, including a previously established Philadelphia translocation. Finally, we demonstrate how some of these variants overlap open chromatin regions, potentially impacting regulatory regions. By integrating both long and short reads, we generated a high-quality reference assembly for eHAP1 cells. The union of long and short reads demonstrates the utility in combining sequencing platforms to generate a high-quality reference genome de novo solely from low coverage data. We expect the resulting eHAP1 genome assembly to provide a useful resource to enable novel experimental applications in this important model cell line.     

A tool for analyzing mate pairs in assemblies (TAMPA).

The current generation of genome assembly programs uses distance and orientation relationships of paired end reads of clones (mate pairs) to order and orient contigs. Mate pair data can also be used to evaluate and compare assemblies after the fact. Earlier work employed a simple heuristic to detect assembly problems by scanning across an assembly to locate peak concentrations of unsatisfied mate pairs. TAMPA is a novel, computational geometry-based approach to detecting assembly breakpoints by exploiting constraints that mate pairs impose on each other. The method can be used to improve assemblies and determine which of two assemblies is correct in the case of sequence disagreement. Results from several human genome assemblies are presented.     

Genome Update. Let the consumer beware: Streptomyces genome sequence quality

A genome sequence assembly represents a model of a genome. This article explores some tools and methods for assessing the quality of an assembly, using publicly available data for Streptomyces species as the example. There is great variability in quality of assemblies deposited in GenBank. Only in a small minority of these assemblies are the raw data available, enabling full appraisal of the assembly quality.