High-throughput sequencing for the identification of binding molecules from DNA-encoded chemical libraries

DNA-encoded chemical libraries are large collections of small organic molecules, individually coupled to DNA fragments that serve as amplifiable identification bar codes. The isolation of specific binders requires a quantitative analysis of the distribution of DNA fragments in the library before and after capture on an immobilized target protein of interest. Here, we show how Illumina sequencing can be applied to the analysis of DNA-encoded chemical libraries, yielding over 10 million DNA sequence tags per flow-lane. The technology can be used in a multiplex format, allowing the encoding and subsequent sequencing of multiple selections in the same experiment. The sequence distributions in DNA-encoded chemical library selections were found to be similar to the ones obtained using 454 technology, thus reinforcing the concept that DNA sequencing is an appropriate avenue for the decoding of library selections. The large number of sequences obtained with the Illumina method now enables the study of very large DNA-encoded chemical libraries (>500,000 compounds) and reduces decoding costs.     

Novel sequencing strategy for repetitive DNA in a Drosophila BAC clone reveals that the centromeric region of the Y chromosome evolved from a telomere

The centromeric and telomeric heterochromatin of eukaryotic chromosomes is mainly composed of middle-repetitive elements, such as transposable elements and tandemly repeated DNA sequences. Because of this repetitive nature, Whole Genome Shotgun Projects have failed in sequencing these regions. We describe a novel kind of transposon-based approach for sequencing highly repetitive DNA sequences in BAC clones. The key to this strategy relies on physical mapping the precise position of the transposon insertion, which enables the correct assembly of the repeated DNA. We have applied this strategy to a clone from the centromeric region of the Y chromosome of Drosophila melanogaster. The analysis of the complete sequence of this clone has allowed us to prove that this centromeric region evolved from a telomere, possibly after a pericentric inversion of an ancestral telocentric chromosome. Our results confirm that the use of transposon-mediated sequencing, including positional mapping information, improves current finishing strategies. The strategy we describe could be a universal approach to resolving the heterochromatic regions of eukaryotic genomes.     

Application of Combined Long Amplicon Sequencing (CoLAS) for Genetic Analysis of Neurofibromatosis Type 1: A Pilot Study

Elaborate analyses of the status of gene mutations in neurofibromatosis type 1 (NF1) are still difficult nowadays due to the large gene sizes, broad mutation spectrum, and the various effects of mutations on mRNA splicing. These problems cannot be solved simply by sequencing the entire coding region using next-generation sequencing (NGS). We recently developed a new strategy, named combined long amplicon sequencing (CoLAS), which is a method for simultaneously analysing the whole genomic DNA region and, also, the full-length cDNA of the disease-causative gene with long-range PCR-based NGS. In this study, CoLAS was specifically arranged for NF1 genetic analysis, then applied to 20 patients (five previously reported and 15 newly recruited patients, including suspicious cases) for optimising the method and to verify its efficacy and benefits. Among new cases, CoLAS detected not only 10 mutations, including three unreported mutations and one mosaic mutation, but also various splicing abnormalities and allelic expression ratios quantitatively. In addition, heterozygous mapping by polymorphisms, including introns, showed copy number monitoring of the entire NF1 gene region was possible in the majority of patients tested. Moreover, it was shown that, when a chromosomal level microdeletion was suspected from heterozygous mapping, it could be detected directly by breakpoint-specific long PCR. In conclusion, CoLAS not simply detect the causative mutation but accurately elucidated the entire structure of the NF1 gene, its mRNA expression, and also the splicing status, which reinforces its high usefulness in the gene analysis of NF1.     

Benefit-of-doubt (BOD) scoring: A sequencing-based method for SNP candidate assessment from high to medium read number data sets

Identification of single nucleotide polymorphisms (SNPs) is a key element in sequence-based genetic analysis. Next generation sequencing offers a cost-effective basis to generate the necessary, large sequence data sets, and bioinformatic methods are being developed to process sequencing machine readouts. We were interested in detection of SNPs in a 350 kb region of an EMS-mutagenized Arabidopsis chromosome 3. The region was selectively analyzed using PCR-generated, overlapping fragments for Solexa sequencing. The ensuing reads provided a high coverage and were processed bioinformatically. In order to assess the SNP candidates obtained with a frequently used alignment program and SNP caller, we developed an additional method that allows the identification of high confidence SNP loci. The method can easily be applied to complete genome sequence data of sufficient coverage.     

Optimization of single-strand conformation polymorphism and sequence analysis of the mitochondrial control region in Pagellus bogaraveo (Sparidae, Teleostei): rationalized tools in fish population biology

We report the isolation and sequencing of 400–550 base pairs (bp) of the mitochondrial DNA (mtDNA) control region of eight species of Sparidae (Perciformes, Teleostei). This sequence information allowed us to design specific primers to one of these species (Pagellus bogaraveo). The new set of primers was used to test a rationalized approach to study the mtDNA nucleotide variability at the intraspecific level. The single-strand conformation polymorphism (SSCP) technique was applied to detect sequence variation in two non-overlapping fragments of the control region of 32 individuals of P. bogaraveo. To assess the sensitivity of the method, the nucleotide sequence of the analysed region was determined for all the specimens. The results showed that, for one of the two fragments, SSCP analysis was able to detect 100% of the underlying genetic variability. In sharp contrast, nucleotide variation of the second DNA fragment was completely unresolved by SSCP under different experimental conditions. This suggests that the resolution power of SSCP is crucially dependent on the nature of the fragment subjected to the analysis; therefore, a preliminary test of the sensitivity of the method should be performed on each specific DNA fragment before starting a large-scale survey. A rationalized approach, combining the SSCP technique and a simplified sequencing procedure, is proposed for studying intraspecific polymorphism at the mtDNA control region in fish.     

A novel method for making nested deletions and its application for sequencing of a 300 kb region of human APP locus.

We developed a novel in vitro method for making nested deletions and applied it to a large-scale DNA sequencing. A DNA fragment to be sequenced (up to 15 kb long) was cloned with a new vector possessing two unique Sfi I sites, digested by Sfi I and ligated to generate a large head-to-tail concatemer. The large concatemer was randomly fragmented by sonication and then redigested by Sfi I to separate insert and vector DNAs. The fragments of various length were then cloned into the other vector(s) specifically designed for selective cloning of insert-derived DNA fragments to generate a library of nested deletions. This method allowed a single person to generate >20 nested deletion libraries sufficient to cover 100 kb in a few days. We applied the method for sequencing of P1 clones and successfully determined the complete sequence of approximately 300 kb of the human amyloid precursor protein (APP) locus on chromosome 21 with a redundancy of 3.8, reasonably low cost and very few gaps remaining to be closed. Development of some new instruments and software is also described which makes this method more applicable for large-scale sequencing.     

Increasing ecological inference from high throughput sequencing of fungi in the environment through a tagging approach

High throughput sequencing methods are widely used in analyses of microbial diversity, but are generally applied to small numbers of samples, which precludes characterization of patterns of microbial diversity across space and time. We have designed a primer-tagging approach that allows pooling and subsequent sorting of numerous samples, which is directed to amplification of a region spanning the nuclear ribosomal internal transcribed spacers and partial large subunit from fungi in environmental samples. To test the method for phylogenetic biases, we constructed a controlled mixture of four taxa representing the Chytridiomycota, Zygomycota, Ascomycota and Basidiomycota. Following cloning and colony restriction fragment length polymorphism analysis, we found no significant difference in representation in 19 of the 23 tested primers. We also generated a clone library from two soil DNA extracts using two primers for each extract and compared 456 clone sequences. Community diversity statistics and contingency table tests applied to counts of operational taxonomic units revealed that the two DNA extracts differed significantly, while the pairs of tagged primers from each extract were indistinguishable. Similar results were obtained using UniFrac phylogenetic comparisons. Together, these results suggest that the pig-tagged primers can be used to increase ecological inference in high throughput sequencing projects on fungi.     

Analysis of the complete human mtDNA genome: methodology and inferences for human evolution

The analysis of mitochondrial DNA (mtDNA) sequences has been a potent tool in our understanding of human evolution. However, almost all studies of human evolution based on mtDNA sequencing have focused on the control region, which constitutes less than 7% of the mitochondrial genome. The rapid development of technology for automated DNA sequencing has made it possible to study the complete mtDNA genomes in large numbers of individuals, opening the field of mitochondrial population genomics. Here we describe a suitable methodology for determining the complete human mitochondrial sequence and the global mtDNA diversity in humans. Also, we discuss the implications of the results with respect to the different hypotheses for the evolution of modern humans.     

化学调控剂对减轻水稻抽穗灌浆期阴害效应的综合评判

应用灰色关联选择受阴害水稻的主要受害指标,以层次分析法和模糊综合评判分析了化学调控剂对减轻水稻在抽穗灌浆期不同时段阴害的效应.结果表明,阴害危害的最大3个指标为结实率、净同化率和根系活性强度;在水稻抽穗后6～20d出现阴害时,喷施化学调控剂的增产效果最明显,隶属度达0.7869.     

Mitochondrial sequence analysis for forensic identification using pyrosequencing technology.

Over recent years, requests for mtDNA analysis in the field of forensic medicine have notably increased, and the results of such analyses have proved to be very useful in forensic cases where nuclear DNA analysis cannot be performed. Traditionally, mtDNA has been analyzed by DNA sequencing of the two hypervariable regions, HVI and HVII, in the D-loop. DNA sequence analysis using the conventional Sanger sequencing is very robust but time consuming and labor intensive. By contrast, mtDNA analysis based on the pyrosequencing technology provides fast and accurate results from the human mtDNA present in many types of evidence materials in forensic casework. The assay has been developed to determine polymorphic sites in the mitochondrial D-loop as well as the coding region to further increase the discrimination power of mtDNA analysis. The pyrosequencing technology for analysis of mtDNA polymorphisms has been tested with regard to sensitivity, reproducibility, and success rate when applied to control samples and actual casework materials. The results show that the method is very accurate and sensitive; the results are easily interpreted and provide a high success rate on casework samples. The panel of pyrosequencing reactions for the mtDNA polymorphisms were chosen to result in an optimal discrimination power in relation to the number of bases determined.     

FOUNTAIN: A JAVA open-source package to assist large sequencing projects

Background  

Better automation, lower cost per reaction and a heightened interest in comparative genomics has led to a dramatic increase in DNA sequencing activities. Although the large sequencing projects of specialized centers are supported by in-house bioinformatics groups, many smaller laboratories face difficulties managing the appropriate processing and storage of their sequencing output. The challenges include documentation of clones, templates and sequencing reactions, and the storage, annotation and analysis of the large number of generated sequences.     

Congruence of ribosomal DNA sequencing, fatty acid methyl ester profiles and morphology for characterization of the genus Rhizophagus (arbuscular mycorrhiza fungus)

Difficulties in obtaining sterile axenic cultures and heterogeneity in nuclear-encoded ribosomal DNA (n-rDNA) sequences within a single arbuscular mycorrhizal spore make genetic analysis of arbuscular mycorrhizal fungi (AMF) a complicated task, and currently available methods of genotyping are inadequate for identification to the species level. Therefore, we applied a multipronged approach on different isolates grown in root organ culture (ROC) belonging to the genus Rhizophagus which were not characterized at species level. Each strain was characterized using the fatty acid methyl ester profile (FAME), partial sequencing of a small subunit-internal transcribed spacer (SSU-ITS) and a large subunit (LSU) region of n-rDNA, and morphological examination of spores. Neighbor-joining trees obtained from the SSU-ITS rDNA sequences were broadly similar to those obtained from the LSU rDNA sequences. FAME profiles of the same isolates used for molecular characterization were obtained using fatty acid datasets, and results were compared to a neighbor-joining tree of n-rDNA sequence. Based on the results of these studues, a combination of morphology, biomarkers (FAME), and molecular sequencing (of highly variable D1-D2 of LSU and ITS) is recommended for phylogenetic analysis and characterization of species/strain of Glomeromycota.     

One-way sequencing of multiple amplicons from tandem repetitive mitochondrial DNA control region

Repetitive DNA sequences not only exist abundantly in eukaryotic nuclear genomes, but also occur as tandem repeats in many animal mitochondrial DNA (mtDNA) control regions. Due to concerted evolution, these repetitive sequences are highly similar or even identical within a genome. When long repetitive regions are the targets of amplification for the purpose of sequencing, multiple amplicons may result if one primer has to be located inside the repeats. Here, we show that, without separating these amplicons by gel purification or cloning, directly sequencing the mitochondrial repeats with the primer outside repetitive region is feasible and efficient. We exemplify it by sequencing the mtDNA control region of the mosquito Aedes albopictus, which harbors typical large tandem DNA repeats. This one-way sequencing strategy is optimal for population surveys.     

Improved reduced representation bisulfite sequencing for epigenomic profiling of clinical samples

Background

DNA methylation plays crucial roles in epigenetic gene regulation in normal development and disease pathogenesis. Efficient and accurate quantification of DNA methylation at single base resolution can greatly advance the knowledge of disease mechanisms and be used to identify potential biomarkers. We developed an improved pipeline based on reduced representation bisulfite sequencing (RRBS) for cost-effective genome-wide quantification of DNA methylation at single base resolution. A selection of two restriction enzymes (Taq^αI and MspI) enables a more unbiased coverage of genomic regions of different CpG densities. We further developed a highly automated software package to analyze bisulfite sequencing results from the Solexa GAIIx system.

Results

With two sequencing lanes, we were able to quantify ~1.8 million individual CpG sites at a minimum sequencing depth of 10. Overall, about 76.7% of CpG islands, 54.9% of CpG island shores and 52.2% of core promoters in the human genome were covered with at least 3 CpG sites per region.

Conclusions

With this new pipeline, it is now possible to perform whole-genome DNA methylation analysis at single base resolution for a large number of samples for understanding how DNA methylation and its changes are involved in development, differentiation, and disease pathogenesis.     

Homozygosity for multiple contiguous single-nucleotide polymorphisms as an indicator of large heterozygous deletions: identification of a novel heterozygous 8-kb intragenic deletion (IVS7-19 to IVS15-17) in a patient with glycogen storage disease type II

Current methods for detection of mutations by polymerase chain reaction (PCR) and sequence analysis frequently are not able to detect heterozygous large deletions. We report the successful use of a novel approach to identify such deletions, based on detection of apparent homozygosity of contiguous single-nucleotide polymorphisms (SNPs). The sequence analysis of genomic DNA PCR products containing all coding exons and flanking introns identified only a single heterozygous mutation (IVS18+2t-->a) in a patient with classic infantile-onset autosomal recessive glycogen storage disease type II (GSDII). Apparent homozygosity for multiple contiguous SNPs detected by this sequencing suggested presence of a large deletion as the second mutation; primers flanking the region of homozygous SNPs permitted identification and characterization by PCR of a large genomic deletion (8.26 kb) extending from IVS7 to IVS15. The data clearly demonstrate the utility of SNPs as markers for large deletions in autosomal recessive diseases when only a single mutation is found, thus complementing currently standard DNA PCR sequence methods for identifying the molecular basis of disease.     

Nucleotide sequence analysis of 95 kb near the 3' end of the murine T-cell receptor alpha/delta chain locus: strategy and methodology.

The nucleotide sequence of a region at the 3' terminus of the murine T-cell receptor alpha/delta chain locus is presented. This region, which encodes the constant region genes for alpha and delta chain polypeptides and all 50 joining gene segments for the alpha chain polypeptide, spans 94,647 bp and includes more than 50 noncoding sequence elements important for T-cell receptor gene rearrangement and expression. DNA sequencing of this region included complete analysis of two cosmid clones and five additional restriction fragments using a random subcloning approach with various manual and automated sequencing strategies. The automated sequencing strategies hold considerable promise for future large-scale DNA sequencing efforts.     

Multiplex random priming of internal restriction fragments for DNA sequencing.

An approach for DNA sequencing is described that circumvents the need for synthetic oligonucleotide primers, which seriously restrict the progress of DNA sequencing in the commonly used protocol. The method is based on the use of short restriction fragments as primers randomly distributed along single-stranded templates. Premapping of target DNA is eliminated and subcloning manipulation is minimized. This method has been used successfully for sequencing genes in the range of 2 kb, for which about 10 restriction fragment primers per kilobase were sufficient to generate a continuous overlapping sequence in alignment. The approach has also been readily applied for an automated sequencing system with the fluorescent chain-terminating dideoxynucleotides, thus implying its potential for sequencing large genomic DNAs.