Euchromatic and heterochromatic compositional properties emerging from the analysis of Solanum lycopersicum BAC sequences

The consortium responsible for the sequencing of the tomato (Solanum lycopersicum) genome initially focused on the sequencing of the euchromatic regions using a BAC-by-BAC strategy. We analyzed the compositional features of the whole collection of BAC sequences publically available. This analysis highlights specific peculiarities of heterochromatic and euchromatic BACs, in particular: the whole BAC collection has i) a large variability in repeat and gene content, ii) a positive and significant correlation of LTR retrotransposons of the Gypsy class with the repeat content and iii) the preferential location of the SINEs (short interspersed nuclear elements) in BAC sequences showing a low repeat content. Our results point out a typical design of the tomato chromosomes and pave the way for further investigations on the relationship between DNA primary structure and chromatin organization in Solanaceae genomes.     

Strategy to sequence the genome of Corynebacterium glutamicum ATCC 13032: use of a cosmid and a bacterial artificial chromosome library

The initial strategy of the Corynebacterium glutamicum genome project was to sequence overlapping inserts of an ordered cosmid library. High-density colony grids of approximately 28 genome equivalents were used for the identification of overlapping clones by Southern hybridization. Altogether 18 contiguous genomic segments comprising 95 overlapping cosmids were assembled. Systematic shotgun sequencing of the assembled cosmid set revealed that only 2.84 Mb (86.6%) of the C. glutamicum genome were represented by the cosmid library. To obtain a complete genome coverage, a bacterial artificial chromosome (BAC) library of the C. glutamicum chromosome was constructed in pBeloBAC11 and used for genome mapping. The BAC library consists of 3168 BACs and represents a theoretical 63-fold coverage of the C. glutamicum genome (3.28 Mb). Southern screening of 2304 BAC clones with PCR-amplified chromosomal markers and subsequent insert terminal sequencing allowed the identification of 119 BACs covering the entire chromosome of C. glutamicum. The minimal set representing a 100% genome coverage contains 44 unique BAC clones with an average overlap of 22 kb. A total of 21 BACs represented linking clones between previously sequenced cosmid contigs and provided a valuable tool for completing the genome sequence of C. glutamicum.     

Combinatorial Pooling Enables Selective Sequencing of the Barley Gene Space

For the vast majority of species – including many economically or ecologically important organisms, progress in biological research is hampered due to the lack of a reference genome sequence. Despite recent advances in sequencing technologies, several factors still limit the availability of such a critical resource. At the same time, many research groups and international consortia have already produced BAC libraries and physical maps and now are in a position to proceed with the development of whole-genome sequences organized around a physical map anchored to a genetic map. We propose a BAC-by-BAC sequencing protocol that combines combinatorial pooling design and second-generation sequencing technology to efficiently approach denovo selective genome sequencing. We show that combinatorial pooling is a cost-effective and practical alternative to exhaustive DNA barcoding when preparing sequencing libraries for hundreds or thousands of DNA samples, such as in this case gene-bearing minimum-tiling-path BAC clones. The novelty of the protocol hinges on the computational ability to efficiently compare hundred millions of short reads and assign them to the correct BAC clones (deconvolution) so that the assembly can be carried out clone-by-clone. Experimental results on simulated data for the rice genome show that the deconvolution is very accurate, and the resulting BAC assemblies have high quality. Results on real data for a gene-rich subset of the barley genome confirm that the deconvolution is accurate and the BAC assemblies have good quality. While our method cannot provide the level of completeness that one would achieve with a comprehensive whole-genome sequencing project, we show that it is quite successful in reconstructing the gene sequences within BACs. In the case of plants such as barley, this level of sequence knowledge is sufficient to support critical end-point objectives such as map-based cloning and marker-assisted breeding.     

Harvesting the mouse genome

The sequencing of the black 6 mouse (strain C57Bl/6) has reached an important juncture. The BAC fingerprint map is almost complete, the BACs have been endsequenced and a seven-fold coverage whole-genome shotgun has been assembled. Now the BAC-by-BAC sequencing phase is under way and in-depth comparative analysis can be carried out on regions that have been the subject of targeted sequencing. This paper reviews the progress so far and looks forward to the promises of finished sequence.     

High throughput direct end sequencing of BAC clones

Libraries constructed in bacterial artificial chromosome (BAC) vectors have become the choice for clone sets in high throughput genomic sequencing projects primarily because of their high stability. BAC libraries have been proposed as a source for minimally over-lapping clones for sequencing large genomic regions, and the use of BAC end sequences (i.e. sequences adjoining the insert sites) has been proposed as a primary means for selecting minimally overlapping clones for sequencing large genomic regions. For this strategy to be effective, high throughput methods for BAC end sequencing of all the clones in deep coverage BAC libraries needed to be developed. Here we describe a low cost, efficient, 96 well procedure for BAC end sequencing. These methods allow us to generate BAC end sequences from human and Arabidoposis libraries with an average read length of >450 bases and with a single pass sequencing average accuracy of >98%. Application of BAC end sequences in genomic sequen-cing is discussed.     

全基因组测序及其在遗传性疾病研究及诊断中的应用

最近,随着测序成本的不断降低,数据分析策略的不断提升,全基因组测序（whole-genome sequencing,WGS）已经在癌症、孟德尔遗传病、复杂疾病的致病基因检测中得到了一定运用,并逐步走向了临床诊断。全基因组测序不但可以检测编码区和非编码区的点突变（SNVs）和插入缺失（InDels）,还可以在全基因组范围内检测拷贝数变异（copy number variation,CNV）以及结构变异（structure variation,SV）。本文详细地介绍了全基因组测序的标准生物信息分析流程与方法,及其在疾病研究、临床诊断中的应用,并对全基因组测序在医学遗传学中的应用与研究进展,以及数据分析方面面临的挑战进行了概述。     

A preprocessor for shotgun assembly of large genomes.

The whole-genome shotgun (WGS) assembly technique has been remarkably successful in efforts to determine the sequence of bases that make up a genome. WGS assembly begins with a large collection of short fragments that have been selected at random from a genome. The sequence of bases at each end of the fragment is determined, albeit imprecisely, resulting in a sequence of letters called a "read." Each letter in a read is assigned a quality value, which estimates the probability that a sequencing error occurred in determining that letter. Reads are typically cut off after about 500 letters, where sequencing errors become endemic. We report on a set of procedures that (1) corrects most of the sequencing errors, (2) changes quality values accordingly, and (3) produces a list of "overlaps," i.e., pairs of reads that plausibly come from overlapping parts of the genome. Our procedures, which we call collectively the "UMD Overlapper," can be run iteratively and as a preprocessor for other assemblers. We tested the UMD Overlapper on Celera's Drosophila reads. When we replaced Celera's overlap procedures in the front end of their assembler, it was able to produce a significantly improved genome.     

Performance comparison of exome DNA sequencing technologies

Whole exome sequencing by high-throughput sequencing of target-enriched genomic DNA (exome-seq) has become common in basic and translational research as a means of interrogating the interpretable part of the human genome at relatively low cost. We present a comparison of three major commercial exome sequencing platforms from Agilent, Illumina and Nimblegen applied to the same human blood sample. Our results suggest that the Nimblegen platform, which is the only one to use high-density overlapping baits, covers fewer genomic regions than the other platforms but requires the least amount of sequencing to sensitively detect small variants. Agilent and Illumina are able to detect a greater total number of variants with additional sequencing. Illumina captures untranslated regions, which are not targeted by the Nimblegen and Agilent platforms. We also compare exome sequencing and whole genome sequencing (WGS) of the same sample, demonstrating that exome sequencing can detect additional small variants missed by WGS.     

Finishing a whole-genome shotgun: release 3 of the Drosophila melanogaster euchromatic genome sequence

Background

The Drosophila melanogaster genome was the first metazoan genome to have been sequenced by the whole-genome shotgun (WGS) method. Two issues relating to this achievement were widely debated in the genomics community: how correct is the sequence with respect to base-pair (bp) accuracy and frequency of assembly errors? And, how difficult is it to bring a WGS sequence to the accepted standard for finished sequence? We are now in a position to answer these questions.

Results

Our finishing process was designed to close gaps, improve sequence quality and validate the assembly. Sequence traces derived from the WGS and draft sequencing of individual bacterial artificial chromosomes (BACs) were assembled into BAC-sized segments. These segments were brought to high quality, and then joined to constitute the sequence of each chromosome arm. Overall assembly was verified by comparison to a physical map of fingerprinted BAC clones. In the current version of the 116.9 Mb euchromatic genome, called Release 3, the six euchromatic chromosome arms are represented by 13 scaffolds with a total of 37 sequence gaps. We compared Release 3 to Release 2; in autosomal regions of unique sequence, the error rate of Release 2 was one in 20,000 bp.

Conclusions

The WGS strategy can efficiently produce a high-quality sequence of a metazoan genome while generating the reagents required for sequence finishing. However, the initial method of repeat assembly was flawed. The sequence we report here, Release 3, is a reliable resource for molecular genetic experimentation and computational analysis.     

Long insert whole genome sequencing for copy number variant and translocation detection

As next-generation sequencing continues to have an expanding presence in the clinic, the identification of the most cost-effective and robust strategy for identifying copy number changes and translocations in tumor genomes is needed. We hypothesized that performing shallow whole genome sequencing (WGS) of 900–1000-bp inserts (long insert WGS, LI-WGS) improves our ability to detect these events, compared with shallow WGS of 300–400-bp inserts. A priori analyses show that LI-WGS requires less sequencing compared with short insert WGS to achieve a target physical coverage, and that LI-WGS requires less sequence coverage to detect a heterozygous event with a power of 0.99. We thus developed an LI-WGS library preparation protocol based off of Illumina’s WGS library preparation protocol and illustrate the feasibility of performing LI-WGS. We additionally applied LI-WGS to three separate tumor/normal DNA pairs collected from patients diagnosed with different cancers to demonstrate our application of LI-WGS on actual patient samples for identification of somatic copy number alterations and translocations. With the evolution of sequencing technologies and bioinformatics analyses, we show that modifications to current approaches may improve our ability to interrogate cancer genomes.     

One Size Doesn't Fit All - RefEditor: Building Personalized Diploid Reference Genome to Improve Read Mapping and Genotype Calling in Next Generation Sequencing Studies

With rapid decline of the sequencing cost, researchers today rush to embrace whole genome sequencing (WGS), or whole exome sequencing (WES) approach as the next powerful tool for relating genetic variants to human diseases and phenotypes. A fundamental step in analyzing WGS and WES data is mapping short sequencing reads back to the reference genome. This is an important issue because incorrectly mapped reads affect the downstream variant discovery, genotype calling and association analysis. Although many read mapping algorithms have been developed, the majority of them uses the universal reference genome and do not take sequence variants into consideration. Given that genetic variants are ubiquitous, it is highly desirable if they can be factored into the read mapping procedure. In this work, we developed a novel strategy that utilizes genotypes obtained a priori to customize the universal haploid reference genome into a personalized diploid reference genome. The new strategy is implemented in a program named RefEditor. When applying RefEditor to real data, we achieved encouraging improvements in read mapping, variant discovery and genotype calling. Compared to standard approaches, RefEditor can significantly increase genotype calling consistency (from 43% to 61% at 4X coverage; from 82% to 92% at 20X coverage) and reduce Mendelian inconsistency across various sequencing depths. Because many WGS and WES studies are conducted on cohorts that have been genotyped using array-based genotyping platforms previously or concurrently, we believe the proposed strategy will be of high value in practice, which can also be applied to the scenario where multiple NGS experiments are conducted on the same cohort. The RefEditor sources are available at https://github.com/superyuan/refeditor.

This is a PLOS Computational Biology Software Article.

     

A high-resolution physical map integrating an anchored chromosome with the BAC physical maps of wheat chromosome 6B

Background

A complete genome sequence is an essential tool for the genetic improvement of wheat. Because the wheat genome is large, highly repetitive and complex due to its allohexaploid nature, the International Wheat Genome Sequencing Consortium (IWGSC) chose a strategy that involves constructing bacterial artificial chromosome (BAC)-based physical maps of individual chromosomes and performing BAC-by-BAC sequencing. Here, we report the construction of a physical map of chromosome 6B with the goal of revealing the structural features of the third largest chromosome in wheat.

Results

We assembled 689 informative BAC contigs (hereafter reffered to as contigs) representing 91 % of the entire physical length of wheat chromosome 6B. The contigs were integrated into a radiation hybrid (RH) map of chromosome 6B, with one linkage group consisting of 448 loci with 653 markers. The order and direction of 480 contigs, corresponding to 87 % of the total length of 6B, were determined. We also characterized the contigs that contained a part of the nucleolus organizer region or centromere based on their positions on the RH map and the assembled BAC clone sequences. Analysis of the virtual gene order along 6B using the information collected for the integrated map revealed the presence of several chromosomal rearrangements, indicating evolutionary events that occurred on chromosome 6B.

Conclusions

We constructed a reliable physical map of chromosome 6B, enabling us to analyze its genomic structure and evolutionary progression. More importantly, the physical map should provide a high-quality and map-based reference sequence that will serve as a resource for wheat chromosome 6B.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1803-y) contains supplementary material, which is available to authorized users.     

BioNano genome mapping of individual chromosomes supports physical mapping and sequence assembly in complex plant genomes

The assembly of a reference genome sequence of bread wheat is challenging due to its specific features such as the genome size of 17 Gbp, polyploid nature and prevalence of repetitive sequences. BAC‐by‐BAC sequencing based on chromosomal physical maps, adopted by the International Wheat Genome Sequencing Consortium as the key strategy, reduces problems caused by the genome complexity and polyploidy, but the repeat content still hampers the sequence assembly. Availability of a high‐resolution genomic map to guide sequence scaffolding and validate physical map and sequence assemblies would be highly beneficial to obtaining an accurate and complete genome sequence. Here, we chose the short arm of chromosome 7D (7DS) as a model to demonstrate for the first time that it is possible to couple chromosome flow sorting with genome mapping in nanochannel arrays and create a de novo genome map of a wheat chromosome. We constructed a high‐resolution chromosome map composed of 371 contigs with an N50 of 1.3 Mb. Long DNA molecules achieved by our approach facilitated chromosome‐scale analysis of repetitive sequences and revealed a ~800‐kb array of tandem repeats intractable to current DNA sequencing technologies. Anchoring 7DS sequence assemblies obtained by clone‐by‐clone sequencing to the 7DS genome map provided a valuable tool to improve the BAC‐contig physical map and validate sequence assembly on a chromosome‐arm scale. Our results indicate that creating genome maps for the whole wheat genome in a chromosome‐by‐chromosome manner is feasible and that they will be an affordable tool to support the production of improved pseudomolecules.     

Comparison of whole‐genome (13X) and capture (87X) resequencing methods for SNP and genotype callings

The number of polymorphisms identified with next‐generation sequencing approaches depends directly on the sequencing depth and therefore on the experimental cost. Although higher levels of depth ensure more sensitive and more specific SNP calls, economic constraints limit the increase of depth for whole‐genome resequencing (WGS). For this reason, capture resequencing is used for studies focusing on only some specific regions of the genome. However, several biases in capture resequencing are known to have a negative impact on the sensitivity of SNP detection. Within this framework, the aim of this study was to compare the accuracy of WGS and capture resequencing on SNP detection and genotype calling, which differ in terms of both sequencing depth and biases. Indeed, we have evaluated the SNP calling and genotyping accuracy in a WGS dataset (13X) and in a capture resequencing dataset (87X) performed on 11 individuals. The percentage of SNPs not identified due to a sevenfold sequencing depth decrease was estimated at 7.8% using a down‐sampling procedure on the capture sequencing dataset. A comparison of the 87X capture sequencing dataset with the WGS dataset revealed that capture‐related biases were leading with the loss of 5.2% of SNPs detected with WGS. Nevertheless, when considering the SNPs detected by both approaches, capture sequencing appears to achieve far better SNP genotyping, with about 4.4% of the WGS genotypes that can be considered as erroneous and even 10% focusing on heterozygous genotypes. In conclusion, WGS and capture deep sequencing can be considered equivalent strategies for SNP detection, as the rate of SNPs not identified because of a low sequencing depth in the former is quite similar to SNPs missed because of method biases of the latter. On the other hand, capture deep sequencing clearly appears more adapted for studies requiring great accuracy in genotyping.     

Optimal pooling for genome re-sequencing with ultra-high-throughput short-read technologies

New generation sequencing technologies offer unique opportunities and challenges for re-sequencing studies. In this article, we focus on re-sequencing experiments using the Solexa technology, based on bacterial artificial chromosome (BAC) clones, and address an experimental design problem. In these specific experiments, approximate coordinates of the BACs on a reference genome are known, and fine-scale differences between the BAC sequences and the reference are of interest. The high-throughput characteristics of the sequencing technology makes it possible to multiplex BAC sequencing experiments by pooling BACs for a cost-effective operation. However, the way BACs are pooled in such re-sequencing experiments has an effect on the downstream analysis of the generated data, mostly due to subsequences common to multiple BACs. The experimental design strategy we develop in this article offers combinatorial solutions based on approximation algorithms for the well-known max n-cut problem and the related max n-section problem on hypergraphs. Our algorithms, when applied to a number of sample cases give more than a 2-fold performance improvement over random partitioning.     

HTS-PEG: A Method for High Throughput Sequencing of the Paired-Ends of Genomic Libraries

Second generation sequencing has been widely used to sequence whole genomes. Though various paired-end sequencing methods have been developed to construct the long scaffold from contigs derived from shotgun sequencing, the classical paired-end sequencing of the Bacteria Artificial Chromosome (BAC) or fosmid libraries by the Sanger method still plays an important role in genome assembly. However, sequencing libraries with the Sanger method is expensive and time-consuming. Here we report a new strategy to sequence the paired-ends of genomic libraries with parallel pyrosequencing, using a Chinese amphioxus (Branchiostoma belcheri) BAC library as an example. In total, approximately 12,670 non-redundant paired-end sequences were generated. Mapping them to the primary scaffolds of Chinese amphioxus, we obtained 413 ultra-scaffolds from 1,182 primary scaffolds, and the N50 scaffold length was increased approximately 55 kb, which is about a 10% improvement. We provide a universal and cost-effective method for sequencing the ultra-long paired-ends of genomic libraries. This method can be very easily implemented in other second generation sequencing platforms.     

An integrated physical, genetic and cytogenetic map of Brachypodium distachyon, a model system for grass research

The pooid subfamily of grasses includes some of the most important crop, forage and turf species, such as wheat, barley and Lolium. Developing genomic resources, such as whole-genome physical maps, for analysing the large and complex genomes of these crops and for facilitating biological research in grasses is an important goal in plant biology. We describe a bacterial artificial chromosome (BAC)-based physical map of the wild pooid grass Brachypodium distachyon and integrate this with whole genome shotgun sequence (WGS) assemblies using BAC end sequences (BES). The resulting physical map contains 26 contigs spanning the 272 Mb genome. BES from the physical map were also used to integrate a genetic map. This provides an independent validation and confirmation of the published WGS assembly. Mapped BACs were used in Fluorescence In Situ Hybridisation (FISH) experiments to align the integrated physical map and sequence assemblies to chromosomes with high resolution. The physical, genetic and cytogenetic maps, integrated with whole genome shotgun sequence assemblies, enhance the accuracy and durability of this important genome sequence and will directly facilitate gene isolation.     

Utilization of a zebra finch BAC library to determine the structure of an avian androgen receptor genomic region

The zebra finch (Taeniopygia guttata) is an important model organism for studying behavior, neuroscience, avian biology, and evolution. To support the study of its genome, we constructed a BAC library (TG__Ba) using DNA from livers of females. The BAC library consists of 147,456 clones with 98% containing inserts of an average size of 134 kb and represents 15.5 haploid genome equivalents. By sequencing a whole BAC, a full-length androgen receptor open reading frame was identified, the first in an avian species. Comparison of BAC end sequences and the whole BAC sequence with the chicken genome draft sequence showed a high degree of conserved synteny between the zebra finch and the chicken genome.