A viral discovery methodology for clinical biopsy samples utilising massively parallel next generation sequencing

Here we describe a virus discovery protocol for a range of different virus genera, that can be applied to biopsy-sized tissue samples. Our viral enrichment procedure, validated using canine and human liver samples, significantly improves viral read copy number and increases the length of viral contigs that can be generated by de novo assembly. This in turn enables the Illumina next generation sequencing (NGS) platform to be used as an effective tool for viral discovery from tissue samples.     

A segment of the apospory-specific genomic region is highly microsyntenic not only between the apomicts Pennisetum squamulatum and buffelgrass, but also with a rice chromosome 11 centromeric-proximal genomic region

Bacterial artificial chromosome (BAC) clones from apomicts Pennisetum squamulatum and buffelgrass (Cenchrus ciliaris), isolated with the apospory-specific genomic region (ASGR) marker ugt197, were assembled into contigs that were extended by chromosome walking. Gene-like sequences from contigs were identified by shotgun sequencing and BLAST searches, and used to isolate orthologous rice contigs. Additional gene-like sequences in the apomicts' contigs were identified by bioinformatics using fully sequenced BACs from orthologous rice contigs as templates, as well as by interspecies, whole-contig cross-hybridizations. Hierarchical contig orthology was rapidly assessed by constructing detailed long-range contig molecular maps showing the distribution of gene-like sequences and markers, and searching for microsyntenic patterns of sequence identity and spatial distribution within and across species contigs. We found microsynteny between P. squamulatum and buffelgrass contigs. Importantly, this approach also enabled us to isolate from within the rice (Oryza sativa) genome contig Rice A, which shows the highest microsynteny and is most orthologous to the ugt197-containing C1C buffelgrass contig. Contig Rice A belongs to the rice genome database contig 77 (according to the current September 12, 2003, rice fingerprint contig build) that maps proximal to the chromosome 11 centromere, a feature that interestingly correlates with the mapping of ASGR-linked BACs proximal to the centromere or centromere-like sequences. Thus, relatedness between these two orthologous contigs is supported both by their molecular microstructure and by their centromeric-proximal location. Our discoveries promote the use of a microsynteny-based positional-cloning approach using the rice genome as a template to aid in constructing the ASGR toward the isolation of genes underlying apospory.     

Clustering exact matches of pairwise sequence alignments by weighted linear regression

Background  

At intermediate stages of genome assembly projects, when a number of contigs have been generated and their validity needs to be verified, it is desirable to align these contigs to a reference genome when it is available. The interest is not to analyze a detailed alignment between a contig and the reference genome at the base level, but rather to have a rough estimate of where the contig aligns to the reference genome, specifically, by identifying the starting and ending positions of such a region. This information is very useful in ordering the contigs, facilitating post-assembly analysis such as gap closure and resolving repeats. There exist programs, such as BLAST and MUMmer, that can quickly align and identify high similarity segments between two sequences, which, when seen in a dot plot, tend to agglomerate along a diagonal but can also be disrupted by gaps or shifted away from the main diagonal due to mismatches between the contig and the reference. It is a tedious and practically impossible task to visually inspect the dot plot to identify the regions covered by a large number of contigs from sequence assembly projects. A forced global alignment between a contig and the reference is not only time consuming but often meaningless.     

Homology-driven assembly of a sequence-ready mouse BAC contig map spanning regions related to the 46-Mb gene-rich euchromatic segments of human chromosome 19

Draft sequence derived from the 46-Mb gene-rich euchromatic portion of human chromosome 19 (HSA19) was utilized to generate a sequence-ready physical map spanning homologous regions of mouse chromosomes. Sequence similarity searches with the human sequence identified more than 1000 individual orthologous mouse genes from which 382 overgo probes were developed for hybridization. Using human gene order and spacing as a model, these probes were used to isolate and assemble bacterial artificial chromosome (BAC) clone contigs spanning homologous mouse regions. Each contig was verified, extended, and joined to neighboring contigs by restriction enzyme fingerprinting analysis. Approximately 3000 mouse BACs were analyzed and assembled into 44 contigs with a combined length of 41.4 Mb. These BAC contigs, covering 90% of HSA19-related mouse DNA, are distributed throughout 15 homology segments derived from different regions of mouse chromosomes 7, 8, 9, 10, and 17. The alignment of the HSA19 map with the ordered mouse BAC contigs revealed a number of structural differences in several overtly conserved homologous regions and more precisely defined the borders of the known regions of HSA19-syntenic homology. Our results demonstrate that given a human draft sequence, BAC contig maps can be constructed quickly for comparative sequencing without the need for preestablished mouse-specific genetic or physical markers and indicate that similar strategies can be applied with equal success to genomes of other vertebrate species.     

Contig selection in physical mapping.

In physical mapping, one orders a set of genetic landmarks or a library of cloned fragments of DNA according to their position in the genome. Our approach to physical mapping divides the problem into smaller and easier subproblems by partitioning the probe set into independent parts (probe contigs). For this purpose we introduce a new distance function between probes, the averaged rank distance (ARD) derived from bootstrap resampling of the raw data. The ARD measures the pairwise distances of probes within a contig and smoothes the distances of probes across different contigs. It shows distinct jumps at contig borders. This makes it appropriate for contig selection by clustering. We have designed a physical mapping algorithm that makes use of these observations and seems to be particularly well suited to the delineation of reliable contigs. We evaluated our method on data sets from two physical mapping projects. On data from the recently sequenced bacterium Xylella fastidiosa, the probe contig set produced by the new method was evaluated using the probe order derived from the sequence information. Our approach yielded a basically correct contig set. On this data we also compared our method to an approach which uses the number of supporting clones to determine contigs. Our map is much more accurate. In comparison to a physical map of Pasteurella haemolytica that was computed using simulated annealing, the newly computed map is considerably cleaner. The results of our method have already proven helpful for the design of experiments aimed at further improving the quality of a map.     

Estimates of conserved microsynteny among the genomes of <Emphasis Type="Italic">Glycine max</Emphasis>, <Emphasis Type="Italic">Medicago truncatula</Emphasis> and <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

A growing body of research indicates that microsynteny is common among dicot genomes. However, most studies focus on just one or a few genomic regions, so the extent of microsynteny across entire genomes remains poorly characterized. To estimate the level of microsynteny between Medicago truncatula (Mt) and Glycine max (soybean), and also among homoeologous segments of soybean, we used a hybridization strategy involving bacterial artificial chromosome (BAC) contigs. A Mt BAC library consisting of 30,720 clones was screened with a total of 187 soybean BAC subclones and restriction fragment length polymorphism (RFLP) probes. These probes came from 50 soybean contig groups, defined as one or more related BAC contigs anchored by the same low-copy probe. In addition, 92 whole soybean BAC clones were hybridized to filters of HindIII-digested Mt BAC DNA to identify additional cases of cross-hybridization after removal of those soybean BACs found to be repetitive in Mt. Microsynteny was inferred when at least two low-copy probes from a single soybean contig hybridized to the same Mt BAC or when a soybean BAC clone hybridized to three or more low-copy fragments from a single Mt BAC. Of the 50 soybean contig groups examined, 54% showed microsynteny to Mt. The degree of conservation among 37 groups of soybean contigs was also investigated. The results indicated substantial conservation among soybean contigs in the same group, with 86.5% of the groups showing at least some level of microsynteny. One contig group was examined in detail by a combination of physical mapping and comparative sequencing of homoeologous segments. A TBLASTX similarity search was performed between 1,085 soybean sequences on the 50 BAC contig groups and the entire Arabidopsis genome. Based on a criterion of sequence homologues <100 kb apart, each with an expected value of < or =1e-07, seven of the 50 soybean contig groups (14%) exhibited microsynteny with Arabidopsis.     

Construction of an ∼2 Mb contig in the region around 80 cM of Arabidopsis thaliana chromosome 2

A method for construction of bacterial artificial chromosome (BAC) contigs from a yeast artifical chromosome (YAC) physical map is described. An ∼2 Mb contig, consisting of two large BAC contigs linked by a small YAC, has been assembled in the region around 80 cM of Arabidopsis thaliana chromosome 2. Clones from this contig will facilitate gene isolation in the region and can be used directly as substrates for DNA sequencing.     

Characterization of three maize bacterial artificial chromosome libraries toward anchoring of the physical map to the genetic map using high-density bacterial artificial chromosome filter hybridization

Three maize (Zea mays) bacterial artificial chromosome (BAC) libraries were constructed from inbred line B73. High-density filter sets from all three libraries, made using different restriction enzymes (HindIII, EcoRI, and MboI, respectively), were evaluated with a set of complex probes including the 185-bp knob repeat, ribosomal DNA, two telomere-associated repeat sequences, four centromere repeats, the mitochondrial genome, a multifragment chloroplast DNA probe, and bacteriophage lambda. The results indicate that the libraries are of high quality with low contamination by organellar and lambda-sequences. The use of libraries from multiple enzymes increased the chance of recovering each region of the genome. Ninety maize restriction fragment-length polymorphism core markers were hybridized to filters of the HindIII library, representing 6x coverage of the genome, to initiate development of a framework for anchoring BAC contigs to the intermated B73 x Mo17 genetic map and to mark the bin boundaries on the physical map. All of the clones used as hybridization probes detected at least three BACs. Twenty-two single-copy number core markers identified an average of 7.4 +/- 3.3 positive clones, consistent with the expectation of six clones. This information is integrated into fingerprinting data generated by the Arizona Genomics Institute to assemble the BAC contigs using fingerprint contig and contributed to the process of physical map construction.     

A bacterial artificial chromosome based physical map of the Ustilago maydis genome.

Ustilago maydis, a basidiomycete, is a model organism among phytopathogenic fungi. A physical map of U. maydis strain 521 was developed from bacterial artificial chromosome (BAC) clones. BAC fingerprints used polyacrylamide gel electrophoresis to separate restriction fragments. Fragments were labeled at the HindIII site and co-digested with HaeIII to reduce fragments to 50-750 bp. Contiguous overlapping sets of clones (contigs) were assembled at nine stringencies (from P < or = 1 x 10(-6) to 1 x 10(-24)). Each assembly nucleated contigs with different percentages of bands overlapping between clones (from 20% to 97%). The number of clones per contig decreased linearly from 41 to 12 from P < or = 1 x 10(-7) to 1 x 10 (-12). The number of separate contigs increased from 56 to 150 over the same range. A hybridization-based physical map of the same BAC clones was compared with the fingerprint contigs built at P < or = 1 x 10(-7). The two methods provided consistent physical maps that were largely validated by genome sequence. The combined hybridization and fingerprint physical map provided a minimum tile path composed of 258 BAC clones (18-20 Mbp) distributed among 28 merged contigs. The genome of U. maydis was estimated to be 20.5 Mbp by pulsed-field gel electrophoresis and 24 Mbp by BAC fingerprints. There were 23 separate chromosomes inferred by both pulsed-field gel electrophoresis and fingerprint contigs. Only 11 of the tile path BAC clones contained recognizable centromere, telomere, and subtelomere repeats (high-copy DNA), suggesting that repeats caused some false merges. There were 247 tile path BAC clones that encompassed about 17.5 Mbp of low-copy DNA sequence. BAC clones are available for repeat and unique gene cluster analysis including tDNA-mediated transformation. Program FingerPrint Contigs maps aligned with each chromosome can be viewed at http://www.siu.edu/~meksem/ustilago_maydis/.     

Fluorescence in situ hybridization mapping of human chromosome 19: mapping and verification of cosmid contigs formed by random restriction enzyme fingerprinting.

Automated restriction enzyme fingerprinting of 7900 cosmids from chromosome 19 and calculation of the likelihood of their overlap based on shared fragments have resulted in the assembly of 743 sets of overlapping cosmids (contigs). We have mapped 22% of the formed contigs (n = 165) and all of the contigs with minimal tiling paths exceeding 6 members (n = 50) to chromosomal bands by fluorescence in situ hybridization using DNA from at least one member cosmid. The estimated average size of the formed contigs is 60-70 kb. Thus, members of a correctly formed contig are expected to lie close to each other in metaphase and interphase chromatin. Therefore, we tested the contig assembly process by comparing the band assignment of two or more members selected from each of 97 contigs. Forty-two of these contigs were further characterized for valid assembly by determining the proximity of members in interphase chromatin. Using these tests, we surveyed a total of 431 joins counted along the minimal tiling path (280 in interphase as well as metaphase) and found 6 erroneous joins, one in each of 6 contigs (6% of tested).     

宏基因组重叠群分箱方法研究综述

宏基因组学技术可以直接从环境中提取微生物的全部遗传物质,而不需要像传统方法一样在培养基上纯培养。这种技术的出现为科学家对微生物群落的结构和功能的认识提供了重要的方法,同时对疾病的诊治、环境的治理以及生命的认识具有重大的意义。从环境中提取出微生物全部遗传物质,对其进行测序从而得到它们的reads片段,通过reads组装工具可以进一步组装成重叠群片段。对重叠群片段进行分箱,可以从宏基因组样本中重建出更多完整的基因。分箱效果的好坏直接影响到后续的生物分析,因此如何将这些含有不同微生物基因混合的重叠群序列进行有效的分箱成为了宏基因组学研究的热点和难点。机器学习方法被广泛应用于宏基因组重叠群分箱,通常分为有监督重叠群分类方法和无监督重叠群聚类方法。该综述针对宏基因组重叠群分箱方法进行了较为全面的阐述,深入剖析了重叠群分类方法与聚类方法,发现其存在分类准确率较低、分箱时间较长、难以从复杂数据集中重建更多微生物基因等问题,并对未来重叠群分箱方法的研究和发展进行了展望。作者建议可以使用半监督学习、集成学习以及深度学习方法,并采用更有效的数据特征表示等途径来提高分箱效果。     

A rapid and accurate strategy for rice contig map construction by combination of fingerprinting and hybridization

A rapid and accurate strategy for rice contig map construction was described. Rice BAC library with average insert of 120 kb in length was used as building materials in contig mapping. The contigs of varied lengths ranging from 500 kb to several megabases with sufficient redundancy to ensure the accuracy of the joining between individual BACs were formed by fingerprinting. The contigs were then assigned to and ordered along the chromosomes by various molecular markers through their hybridization against the whole rice genomic library. The accuracy of clone overlaps in contig was further confirmed by the existence in contigs of well fit stacks of marker-lodged clones. He contigs thus obtained covered nearly the rice genome.     

Evidence that the separation of Mhc class II from class I loci in the zebrafish,Danio rerio,occurred by translocation

In the zebrafish, Danio rerio, and other teleosts, the class I and class II loci of the major histocompatibility complex ( Mhc) reside on different chromosomes. To shed light on the events that might have generated this difference from tetrapods, in which these two types of loci are clustered in a single chromosomal region, the organization of the class II loci in linkage group 8 of the zebrafish was determined by the characterization of contigs of PAC clones. Three contigs were defined: DAB, DCB, and DBB. The 350-kb-long DAB contig contained only four genes: DDB, DAB, SLC7A4, and DAA. The 150-kb-long DCB contig contained the DCB, DCA, and fz10 genes at an undetermined distance from the DAB contig. And the 120-kb-long DBB contig comprised the DBB gene presumably in another linkage group. The low gene density of the linkage group 8 contigs, contrasting with the high gene density of the zebrafish class I region, and the close association with genes [ SLC7A4 coding for an amino acid transporter, and fz10 (frizzled 10) coding for a receptor of the WNT glycoprotein] that are not linked with the tetrapod Mhc, is interpreted to mean that the separation of the class II from class I loci in teleosts occurred by translocation rather than by genomic or chromosomal duplication.     

Evaluation of a cosmid contig physical map of human chromosome 16.

A cosmid contig physical map of human chromosome 16 has been developed by repetitive sequence finger-printing of approximately 4000 cosmid clones obtained from a chromosome 16-specific cosmid library. The arrangement of clones in contigs is determined by (1) estimating cosmid length and determining the likelihoods for all possible pairwise clone overlaps, using the fingerprint data, and (2) using an optimization technique to fit contig maps to these estimates. Two important questions concerning this contig map are how much of chromosome 16 is covered and how accurate are the assembled contigs. Both questions can be addressed by hybridization of single-copy sequence probes to gridded arrays of the cosmids. All of the fingerprinted clones have been arrayed on nylon membranes so that any region of interest can be identified by hybridization. The hybridization experiments indicate that approximately 84% of the euchromatic arms of chromosome 16 are covered by contigs and singleton cosmids. Both grid hybridization (26 contigs) and pulsed-field gel electrophoresis experiments (11 contigs) confirmed the assembled contigs, indicating that false positive overlaps occur infrequently in the present map. Furthermore, regional localization of 93 contigs and singleton cosmids to a somatic cell hybrid mapping panel indicates that there is no bias in the coverage of the euchromatic arms.     

A BAC-based physical map of the channel catfish genome

Catfish is the major aquaculture species in the United States. To enhance its genome studies involving genetic linkage and comparative mapping, a bacterial artificial chromosome (BAC) contig-based physical map of the channel catfish (Ictalurus punctatus) genome was generated using four-color fluorescence-based fingerprints. Fingerprints of 34,580 BAC clones (5.6x genome coverage) were generated for the FPC assembly of the BAC contigs. A total of 3307 contigs were assembled using a cutoff value of 1x10(-20). Each contig contains an average of 9.25 clones with an average size of 292 kb. The combined contig size for all contigs was 0.965 Gb, approximately the genome size of the channel catfish. The reliability of the contig assembly was assessed by both hybridization of gene probes to BAC clones contained in the fingerprinted assembly and validation of randomly selected contigs using overgo probes designed from BAC end sequences. The presented physical map should greatly enhance genome research in the catfish, particularly aiding in the identification of genomic regions containing genes underlying important performance traits.     

Characterization of a new BAC library for rainbow trout: evidence for multi-locus duplication

A 10X rainbow trout bacterial artificial chromosome (BAC) library was constructed to aid in the physical and genetic mapping efforts of the rainbow trout genome. The library was derived from the Swanson clonal line (YY male) and consists of 184,704 clones with an average insert size of 137,500 bp (PFGE) or 118,700 bp (DNA fingerprinting). The clones were gridded onto 10 large nylon membranes to produce high-density arrays for screening the library by hybridization. The library was probed with 11 cDNAs from the NCCCWA EST project chosen because of interest in their homology to known gene sequences, seven known genes, and a Y-specific sex marker. Putative positive clones identified by hybridization were re-arrayed and gridded for secondary confirmation. FPC analysis of HindIII and EcoRV DNA fingerprinting was used to estimate the level of redundancy in the library, to construct BAC contigs and to detect duplicated loci in the semi-duplicated rainbow trout genome. A good correlation (R2 = 0.7) was found between the number of hits per probe and the number of contigs that were assembled from the positive BACs. The average number of BACs per contig was 9.6, which is in good agreement with 10X genome coverage of the library. Two-thirds of the loci screened were predicted to be duplicated as the positive BACs for those genes were assembled into two or three different contigs, which suggests that most of the rainbow trout genome is duplicated.     

Bacterial genome mapper: A comparative bacterial genome mapping tool

Recently, next generation sequencing (NGS) technologies have led to a revolutionary increase in sequencing speed and costefficacy. Consequently, a vast number of contigs from many recently sequenced bacterial genomes remain to be accurately mapped and annotated, requiring the development of more convenient bioinformatics programs. In this paper, we present a newly developed web-based bioinformatics program, Bacterial Genome Mapper, which is suitable for mapping and annotating contigs that have been assembled from bacterial genome sequence raw data. By constructing a multiple alignment map between target contig sequences and two reference bacterial genome sequences, this program also provides very useful comparative genomics analysis of draft bacterial genomes. AVAILABILITY: The database is available for free at http://mbgm.kribb.re.kr.     

A BAC-based physical map of Zhikong scallop (Chlamys farreri Jones et Preston)

Zhikong scallop (Chlamys farreri) is one of the most economically important aquaculture species in China. Physical maps are crucial tools for genome sequencing, gene mapping and cloning, genetic improvement and selective breeding. In this study, we have developed a genome-wide, BAC-based physical map for the species. A total of 81,408 clones from two BAC libraries of the scallop were fingerprinted using an ABI 3130xl Genetic Analyzer and a fingerprinting kit developed in our laboratory. After data processing, 63,641 (～5.8× genome coverage) fingerprints were validated and used in the physical map assembly. A total of 3,696 contigs were assembled for the physical map. Each contig contained an average of 10.0 clones, with an average physical size of 490 kb. The combined total physical size of all contigs was 1.81 Gb, equivalent to approximately 1.5 fold of the scallop haploid genome. A total of 10,587 BAC end sequences (BESs) and 167 markers were integrated into the physical map. We evaluated the physical map by overgo hybridization, BAC-FISH (fluorescence in situ hybridization), contig BAC pool screening and source BAC library screening. The results have provided evidence of the high reliability of the contig physical map. This is the first physical map in mollusc; therefore, it provides an important platform for advanced research of genomics and genetics, and mapping of genes and QTL of economical importance, thus facilitating the genetic improvement and selective breeding of the scallop and other marine molluscs.     

Comparison between a coffee single copy chromosomal region and Arabidopsis duplicated counterparts evidenced high level synteny between the coffee genome and the ancestral Arabidopsis genome

The Arabidopsis thaliana genome sequence provides a catalogue of reference genes that can be used for comparative analysis of other species thereby facilitating map-based cloning in economically important crops. We made use of a coffee bacterial artificial chromosome (BAC) contig linked to the S_H3 leaf rust resistance gene to assess microsynteny between coffee (Coffea arabica L.) and Arabidopsis. Microsynteny was revealed and the matching counterparts to C. arabica contigs were seen to be scattered throughout four different syntenic segments of Arabidopsis on chromosomes (Ath) I, III, IV and V. Coffee BAC filter hybridizations were performed using coffee putative conserved orthologous sequences to Arabidopsis predicted genes located on the different Arabidopsis syntenic regions. The coffee BAC contig related to the S_H3 region was successfully consolidated and later on validated by fingerprinting. Furthermore, the anchoring markers appeared in same order on the coffee BAC contigs and in all Arabidopsis segments with the exception of a single inversion on AtIII and AtIV Arabidopsis segments. However, the S_H3 coffee region appears to be closer to the ancestral genome segment (before the divergence of Arabidopsis and coffee) than any of the duplicated counterparts in the present-day Arabidopsis genome. The genome duplication events at the origin of its Arabidopsis counterparts occurred most probably after the separation (i.e. 94 million years ago) of Euasterid (Coffee) and Eurosid (Arabidopsis).     

Analysis of expressed sequence tags from the fungus Aspergillus oryzae cultured under different conditions.

We performed random sequencing of cDNAs from nine biologically or industrially important cultures of the industrially valuable fungus Aspergillus oryzae to obtain expressed sequence tags (ESTs). Consequently, 21 446 raw ESTs were accumulated and subsequently assembled to 7589 non-redundant consensus sequences (contigs). Among all contigs, 5491 (72.4%) were derived from only a particular culture. These included 4735 (62.4%) singletons, i.e. lone ESTs overlapping with no others. These data showed that consideration of culture grown under various conditions as cDNA sources enabled efficient collection of ESTs. BLAST searches against the public databases showed that 2953 (38.9%) of the EST contigs showed significant similarities to deposited sequences with known functions, 793 (10.5%) were similar to hypothetical proteins, and the remaining 3843 (50.6%) showed no significant similarity to sequences in the databases. Culture-specific contigs were extracted on the basis of the EST frequency normalized by the total number for each culture condition. In addition, contig sequences were compared with sequence sets in eukaryotic orthologous groups (KOGs), and classified into the KOG functional categories.