Direct mutation analysis by high-throughput sequencing: from germline to low-abundant, somatic variants

DNA mutations are the source of genetic variation within populations. The majority of mutations with observable effects are deleterious. In humans mutations in the germ line can cause genetic disease. In somatic cells multiple rounds of mutations and selection lead to cancer. The study of genetic variation has progressed rapidly since the completion of the draft sequence of the human genome. Recent advances in sequencing technology, most importantly the introduction of massively parallel sequencing (MPS), have resulted in more than a hundred-fold reduction in the time and cost required for sequencing nucleic acids. These improvements have greatly expanded the use of sequencing as a practical tool for mutation analysis. While in the past the high cost of sequencing limited mutation analysis to selectable markers or small forward mutation targets assumed to be representative for the genome overall, current platforms allow whole genome sequencing for less than $5000. This has already given rise to direct estimates of germline mutation rates in multiple organisms including humans by comparing whole genome sequences between parents and offspring. Here we present a brief history of the field of mutation research, with a focus on classical tools for the measurement of mutation rates. We then review MPS, how it is currently applied and the new insight into human and animal mutation frequencies and spectra that has been obtained from whole genome sequencing. While great progress has been made, we note that the single most important limitation of current MPS approaches for mutation analysis is the inability to address low-abundance mutations that turn somatic tissues into mosaics of cells. Such mutations are at the basis of intra-tumor heterogeneity, with important implications for clinical diagnosis, and could also contribute to somatic diseases other than cancer, including aging. Some possible approaches to gain access to low-abundance mutations are discussed, with a brief overview of new sequencing platforms that are currently waiting in the wings to advance this exploding field even further.     

Massively Parallel Sequencing of Patients with Intellectual Disability,Congenital Anomalies and/or Autism Spectrum Disorders with a Targeted Gene Panel

Developmental delay and/or intellectual disability (DD/ID) affects 1–3% of all children. At least half of these are thought to have a genetic etiology. Recent studies have shown that massively parallel sequencing (MPS) using a targeted gene panel is particularly suited for diagnostic testing for genetically heterogeneous conditions. We report on our experiences with using massively parallel sequencing of a targeted gene panel of 355 genes for investigating the genetic etiology of eight patients with a wide range of phenotypes including DD/ID, congenital anomalies and/or autism spectrum disorder. Targeted sequence enrichment was performed using the Agilent SureSelect Target Enrichment Kit and sequenced on the Illumina HiSeq2000 using paired-end reads. For all eight patients, 81–84% of the targeted regions achieved read depths of at least 20×, with average read depths overlapping targets ranging from 322× to 798×. Causative variants were successfully identified in two of the eight patients: a nonsense mutation in the ATRX gene and a canonical splice site mutation in the L1CAM gene. In a third patient, a canonical splice site variant in the USP9X gene could likely explain all or some of her clinical phenotypes. These results confirm the value of targeted MPS for investigating DD/ID in children for diagnostic purposes. However, targeted gene MPS was less likely to provide a genetic diagnosis for children whose phenotype includes autism.     

Chloroplast genome sequences from total DNA for plant identification

Chloroplast DNA sequence data are a versatile tool for plant identification or barcoding and establishing genetic relationships among plant species. Different chloroplast loci have been utilized for use at close and distant evolutionary distances in plants, and no single locus has been identified that can distinguish between all plant species. Advances in DNA sequencing technology are providing new cost‐effective options for genome comparisons on a much larger scale. Universal PCR amplification of chloroplast sequences or isolation of pure chloroplast fractions, however, are non‐trivial. We now propose the analysis of chloroplast genome sequences from massively parallel sequencing (MPS) of total DNA as a simple and cost‐effective option for plant barcoding, and analysis of plant relationships to guide gene discovery for biotechnology. We present chloroplast genome sequences of five grass species derived from MPS of total DNA. These data accurately established the phylogenetic relationships between the species, correcting an apparent error in the published rice sequence. The chloroplast genome may be the elusive single‐locus DNA barcode for plants.     

Differentiating between monozygotic twins through next-generation mitochondrial genome sequencing

Monozygotic (MZ) twins, considered to be genetically identical, cannot be distinguished from one another by standard forensic DNA testing. A recent study employed whole genome sequencing to identify extremely rare mutations and reported that mutation analysis could be used to differentiate between MZ twins. Compared with nuclear DNA, mitochondrial DNA (mtDNA) has higher mutation rates; therefore, minor differences theoretically exist in MZ twins' mitochondrial genome (mtGenome). However, conventional Sanger-type sequencing (STS) is neither amenable to, nor feasible for, the detection of low-level sequence variants. The recent introduction of massively parallel sequencing (MPS) has the capability to sequence many targeted regions of multiple samples simultaneously with desirable depth of coverage. Thus, the aim of this study was to assess whether full mtGenome sequencing analysis can be used to differentiate between MZ twins. Ten sets of MZ twins provided blood samples that underwent extraction, quantification, mtDNA enrichment, library preparation, and ultra-deep sequencing. Point heteroplasmies were observed in eight sets of MZ twins, and a single nucleotide variant (nt15301) was detected in five sets of MZ twins. Thus, this study demonstrates that ultra-deep mtGenome sequencing could be used to differentiate between MZ twins.     

CSYseq: The first Y-chromosome sequencing tool typing a large number of Y-SNPs and Y-STRs to unravel worldwide human population genetics

Male-specific Y-chromosome (chrY) polymorphisms are interesting components of the DNA for population genetics. While single nucleotide polymorphisms (Y-SNPs) indicate distant evolutionary ancestry, short tandem repeats (Y-STRs) are able to identify close familial kinships. Detailed chrY analysis provides thus both biogeographical background information as paternal lineage identification. The rapid advancement of high-throughput massive parallel sequencing (MPS) technology in the past decade has revolutionized genetic research. Using MPS, single-base information of both Y-SNPs as Y-STRs can be analyzed in a single assay typing multiple samples at once. In this study, we present the first extensive chrY-specific targeted resequencing panel, the ‘CSYseq’, which simultaneously identifies slow mutating Y-SNPs as evolution markers and rapid mutating Y-STRs as patrilineage markers. The panel was validated by paired-end sequencing of 130 males, distributed over 65 deep-rooted pedigrees covering 1,279 generations. The CSYseq successfully targets 15,611 Y-SNPs including 9,014 phylogenetic informative Y-SNPs to identify 1,443 human evolutionary Y-subhaplogroup lineages worldwide. In addition, the CSYseq properly targets 202 Y-STRs, including 81 slow, 68 moderate, 27 fast and 26 rapid mutating Y-STRs to individualize close paternal relatives. The targeted chrY markers cover a high average number of reads (Y-SNP = 717, Y-STR = 150), easy interpretation, powerful discrimination capacity and chrY specificity. The CSYseq is interesting for research on different time scales: to identify evolutionary ancestry, to find distant family and to discriminate closely related males. Therefore, this panel serves as a unique tool valuable for a wide range of genetic-genealogical applications in interdisciplinary research within evolutionary, population, molecular, medical and forensic genetics.     

A Single Cell Level Based Method for Copy Number Variation Analysis by Low Coverage Massively Parallel Sequencing

Copy number variations (CNVs), a common genomic mutation associated with various diseases, are important in research and clinical applications. Whole genome amplification (WGA) and massively parallel sequencing have been applied to single cell CNVs analysis, which provides new insight for the fields of biology and medicine. However, the WGA-induced bias significantly limits sensitivity and specificity for CNVs detection. Addressing these limitations, we developed a practical bioinformatic methodology for CNVs detection at the single cell level using low coverage massively parallel sequencing. This method consists of GC correction for WGA-induced bias removal, binary segmentation algorithm for locating CNVs breakpoints, and dynamic threshold determination for final signals filtering. Afterwards, we evaluated our method with seven test samples using low coverage sequencing (4∼9.5%). Four single-cell samples from peripheral blood, whose karyotypes were confirmed by whole genome sequencing analysis, were acquired. Three other test samples derived from blastocysts whose karyotypes were confirmed by SNP-array analysis were also recruited. The detection results for CNVs of larger than 1 Mb were highly consistent with confirmed results reaching 99.63% sensitivity and 97.71% specificity at base-pair level. Our study demonstrates the potential to overcome WGA-bias and to detect CNVs (>1 Mb) at the single cell level through low coverage massively parallel sequencing. It highlights the potential for CNVs research on single cells or limited DNA samples and may prove as a promising tool for research and clinical applications, such as pre-implantation genetic diagnosis/screening, fetal nucleated red blood cells research and cancer heterogeneity analysis.     

Rapid pulsed whole genome sequencing for comprehensive acute diagnostics of inborn errors of metabolism

Background

Massively parallel DNA sequencing (MPS) has the potential to revolutionize diagnostics, in particular for monogenic disorders. Inborn errors of metabolism (IEM) constitute a large group of monogenic disorders with highly variable clinical presentation, often with acute, nonspecific initial symptoms. In many cases irreversible damage can be reduced by initiation of specific treatment, provided that a correct molecular diagnosis can be rapidly obtained. MPS thus has the potential to significantly improve both diagnostics and outcome for affected patients in this highly specialized area of medicine.

Results

We have developed a conceptually novel approach for acute MPS, by analysing pulsed whole genome sequence data in real time, using automated analysis combined with data reduction and parallelization. We applied this novel methodology to an in-house developed customized work flow enabling clinical-grade analysis of all IEM with a known genetic basis, represented by a database containing 474 disease genes which is continuously updated. As proof-of-concept, two patients were retrospectively analysed in whom diagnostics had previously been performed by conventional methods. The correct disease-causing mutations were identified and presented to the clinical team after 15 and 18 hours from start of sequencing, respectively. With this information available, correct treatment would have been possible significantly sooner, likely improving outcome.

Conclusions

We have adapted MPS to fit into the dynamic, multidisciplinary work-flow of acute metabolic medicine. As the extent of irreversible damage in patients with IEM often correlates with timing and accuracy of management in early, critical disease stages, our novel methodology is predicted to improve patient outcome. All procedures have been designed such that they can be implemented in any technical setting and to any genetic disease area. The strategy conforms to international guidelines for clinical MPS, as only validated disease genes are investigated and as clinical specialists take responsibility for translation of results. As follow-up in patients without any known IEM, filters can be lifted and the full genome investigated, after genetic counselling and informed consent.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1090) contains supplementary material, which is available to authorized users.     

Next generation sequencing in cardiomyopathy: towards personalized genomics and medicine

Next generation sequencing (NGS) is perhaps one of the most exciting advances in the field of life sciences and biomedical research in the last decade. With the availability of massive parallel sequencing, human DNA blueprint can be decoded to explore the hidden information with reduced time and cost. This technology has been used to understand the genetic aspects of various diseases including cardiomyopathies. Mutations for different cardiomyopathies have been identified and cataloging mutations on phenotypic basis are underway and are expected to lead to new discoveries that may translate to novel diagnostic, prognostic and therapeutic targets. With ease in handling NGS, cost effectiveness and fast data output, NGS is now considered as a diagnostic tool for cardiomyopathy by providing targeted gene sequencing. In addition to the number of genetic variants that are identified in cardiomyopathies, there is a need of quicker and easy way to screen multiple genes associated with the disease. In this review, an attempt has been made to explain the NGS technology, methods and applications in cardiomyopathies and their perspective in clinical practice and challenges which are to be addressed.     

Microbial forensics: new breakthroughs and future prospects

Applied Microbiology and Biotechnology - Recent advances in genetic data generation, through massive parallel sequencing (MPS), storage and analysis have fostered significant progresses in...     

Next generation molecular diagnosis of mitochondrial disorders

Mitochondrial disorders are by far the most genetically heterogeneous group of diseases, involving two genomes, the 16.6 kb mitochondrial genome and ~ 1500 genes encoded in the nuclear genome. For maternally inherited mitochondrial DNA disorders, a complete molecular diagnosis requires several different methods for the detection and quantification of mtDNA point mutations and large deletions. For mitochondrial disorders caused by autosomal recessive, dominant, and X-linked nuclear genes, the diagnosis has relied on clinical, biochemical, and molecular studies to point to a group of candidate genes followed by stepwise Sanger sequencing of the candidate genes one-by-one. The development of Next Generation Sequencing (NGS) has revolutionized the diagnostic approach. Using massively parallel sequencing (MPS) analysis of the entire mitochondrial genome, mtDNA point mutations and deletions can be detected and quantified in one single step. The NGS approach also allows simultaneous analyses of a group of genes or the whole exome, thus, the mutations in causative gene(s) can be identified in one-step. New approaches make genetic analyses much faster and more efficient. Huge amounts of sequencing data produced by the new technologies brought new challenges to bioinformatics, analytical pipelines, and interpretation of numerous novel variants. This article reviews the clinical utility of next generation sequencing for the molecular diagnoses of complex dual genome mitochondrial disorders.     

A novel assembly pipeline and functional annotations for targeted sequencing: A case study on the globally threatened Margaritiferidae (Bivalvia: Unionida)

The proliferation of genomic sequencing approaches has significantly impacted the field of phylogenetics. Target capture approaches provide a cost-effective, fast and easily applied strategy for phylogenetic inference of non-model organisms. However, several existing target capture processing pipelines are incapable of incorporating whole genome sequencing (WGS). Here, we develop a new pipeline for capture and de novo assembly of the targeted regions using whole genome re-sequencing reads. This new pipeline captured targeted loci accurately, and given its unbiased nature, can be used with any target capture probe set. Moreover, due to its low computational demand, this new pipeline may be ideal for users with limited resources and when high-coverage sequencing outputs are required. We demonstrate the utility of our approach by incorporating WGS data into the first comprehensive phylogenomic reconstruction of the freshwater mussel family Margaritiferidae. We also provide a catalogue of well-curated functional annotations of these previously uncharacterized freshwater mussel-specific target regions, representing a complementary tool for scrutinizing phylogenetic inferences while expanding future applications of the probe set.     

HOMER2, a Stereociliary Scaffolding Protein,Is Essential for Normal Hearing in Humans and Mice

Hereditary hearing loss is a clinically and genetically heterogeneous disorder. More than 80 genes have been implicated to date, and with the advent of targeted genomic enrichment and massively parallel sequencing (TGE+MPS) the rate of novel deafness-gene identification has accelerated. Here we report a family segregating post-lingual progressive autosomal dominant non-syndromic hearing loss (ADNSHL). After first excluding plausible variants in known deafness-causing genes using TGE+MPS, we completed whole exome sequencing in three hearing-impaired family members. Only a single variant, p.Arg185Pro in HOMER2, segregated with the hearing-loss phenotype in the extended family. This amino acid change alters a highly conserved residue in the coiled-coil domain of HOMER2 that is essential for protein multimerization and the HOMER2-CDC42 interaction. As a scaffolding protein, HOMER2 is involved in intracellular calcium homeostasis and cytoskeletal organization. Consistent with this function, we found robust expression in stereocilia of hair cells in the murine inner ear and observed that over-expression of mutant p.Pro185 HOMER2 mRNA causes anatomical changes of the inner ear and neuromasts in zebrafish embryos. Furthermore, mouse mutants homozygous for the targeted deletion of Homer2 present with early-onset rapidly progressive hearing loss. These data provide compelling evidence that HOMER2 is required for normal hearing and that its sequence alteration in humans leads to ADNSHL through a dominant-negative mode of action.     

Prenatal detection of aneuploidy and imbalanced chromosomal arrangements by massively parallel sequencing

Fetal chromosomal abnormalities are the most common reasons for invasive prenatal testing. Currently, G-band karyotyping and several molecular genetic methods have been established for diagnosis of chromosomal abnormalities. Although these testing methods are highly reliable, the major limitation remains restricted resolutions or can only achieve limited coverage on the human genome at one time. The massively parallel sequencing (MPS) technologies which can reach single base pair resolution allows detection of genome-wide intragenic deletions and duplication challenging karyotyping and microarrays as the tool for prenatal diagnosis. Here we reported a novel and robust MPS-based method to detect aneuploidy and imbalanced chromosomal arrangements in amniotic fluid (AF) samples. We sequenced 62 AF samples on Illumina GAIIx platform and with averagely 0.01× whole genome sequencing data we detected 13 samples with numerical chromosomal abnormalities by z-test. With up to 2× whole genome sequencing data we were able to detect microdeletion/microduplication (ranged from 1.4 Mb to 37.3 Mb of 5 samples from chorionic villus sampling (CVS) using SeqSeq algorithm. Our work demonstrated MPS is a robust and accurate approach to detect aneuploidy and imbalanced chromosomal arrangements in prenatal samples.     

Application of Massively Parallel Sequencing to Genetic Diagnosis in Multiplex Families with Idiopathic Sensorineural Hearing Impairment

Despite the clinical utility of genetic diagnosis to address idiopathic sensorineural hearing impairment (SNHI), the current strategy for screening mutations via Sanger sequencing suffers from the limitation that only a limited number of DNA fragments associated with common deafness mutations can be genotyped. Consequently, a definitive genetic diagnosis cannot be achieved in many families with discernible family history. To investigate the diagnostic utility of massively parallel sequencing (MPS), we applied the MPS technique to 12 multiplex families with idiopathic SNHI in which common deafness mutations had previously been ruled out. NimbleGen sequence capture array was designed to target all protein coding sequences (CDSs) and 100 bp of the flanking sequence of 80 common deafness genes. We performed MPS on the Illumina HiSeq2000, and applied BWA, SAMtools, Picard, GATK, Variant Tools, ANNOVAR, and IGV for bioinformatics analyses. Initial data filtering with allele frequencies (<5% in the 1000 Genomes Project and 5400 NHLBI exomes) and PolyPhen2/SIFT scores (>0.95) prioritized 5 indels (insertions/deletions) and 36 missense variants in the 12 multiplex families. After further validation by Sanger sequencing, segregation pattern, and evolutionary conservation of amino acid residues, we identified 4 variants in 4 different genes, which might lead to SNHI in 4 families compatible with autosomal dominant inheritance. These included GJB2 p.R75Q, MYO7A p.T381M, KCNQ4 p.S680F, and MYH9 p.E1256K. Among them, KCNQ4 p.S680F and MYH9 p.E1256K were novel. In conclusion, MPS allows genetic diagnosis in multiplex families with idiopathic SNHI by detecting mutations in relatively uncommon deafness genes.     

Characterizing homologues of crop domestication genes in poorly described wild relatives by high‐throughput sequencing of whole genomes

Wild crop relatives represent a source of novel alleles for crop genetic improvement. Screening biodiversity for useful or diverse gene homologues has often been based upon the amplification of targeted genes using available sequence information to design primers that amplify the target gene region across species. The crucial requirement of this approach is the presence of sequences with sufficient conservation across species to allow for the design of universal primers. This approach is often not successful with diverse organisms or highly variable genes. Massively parallel sequencing (MPS) can quickly produce large amounts of sequence data and provides a viable option for characterizing homologues of known genes in poorly described genomes. MPS of genomic DNA was used to obtain species‐specific sequence information for 18 rice genes related to domestication characteristics in a wild relative of rice, Microlaena stipoides. Species‐specific primers were available for 16 genes compared with 12 genes using the universal primer method. The use of species‐specific primers had the potential to cover 92% of the sequence of these genes, while traditional universal primers could only be designed to cover 80%. A total of 24 species‐specific primer pairs were used to amplify gene homologues, and 11 primer pairs were successful in capturing six gene homologues. The 23 million, 36‐base pair (bp) paired end reads, equated to an average of 2X genome coverage, facilitated the successful amplification and sequencing of six target gene homologues, illustrating an important approach to the discovery of useful genes in wild crop relatives.     

Preparation of genome-wide DNA fragment libraries using bisulfite in polyacrylamide gel electrophoresis slices with formamide denaturation and quality control for massively parallel sequencing by oligonucleotide ligation and detection

Bisulfite sequencing is widely used for analysis of DNA methylation status (i.e., 5-methylcytosine [5mC] vs. cytosine [C]) in CpG-rich or other loci in genomic DNA (gDNA). Such methods typically involve reaction of gDNA with bisulfite followed by polymerase chain reaction (PCR) amplification of specific regions of interest that, overall, converts C→T (thymine) and 5mC→C and then capillary sequencing to measure C versus T composition at CpG sites. Massively parallel sequencing by oligonucleotide ligation and detection (SOLiD) has recently enabled relatively low-cost whole genome sequencing, and it would be highly desirable to apply such massively parallel sequencing to bisulfite-converted whole genomes to determine DNA methylation status of an entire genome, which has heretofore not been reported. As an initial step toward achieving this goal, we have extended our ongoing interest in improving bisulfite conversion sample preparation to include a human genome-wide fragment library for SOliD. The current article features novel use of formamide denaturant during bisulfite conversion of a suitably constructed library directly in a band slice from polyacryamide gel electrophoresis (PAGE). To validate this new protocol for 5mC-protected fragment library conversion, which we refer to as Bis-PAGE, capillary-based size analysis and Sanger sequencing were carried out for individual amplicons derived from single-molecule PCR (smPCR) of randomly selected library fragments. smPCR/Capillary Sanger sequencing of approximately 200 amplicons unambiguously demonstrated greater than 99% C→T conversion. All of these approximately 200 Sanger sequences were analyzed with a previously published web-accessible bioinformatics tool (methBLAST) for mapping to human chromosomes, the results of which indicated random distribution of analyzed fragments across all chromosomes. Although these particular Bis-PAGE conversion and quality control methods were exemplified in the context of a fragment library for SOLiD, the concepts can be generalized to include other genome-wide library constructions intended for DNA methylation analysis by alternative high-throughput or massively parallelized methods that are currently available.     

Comparison of Pre-Analytical FFPE Sample Preparation Methods and Their Impact on Massively Parallel Sequencing in Routine Diagnostics

Over the last years, massively parallel sequencing has rapidly evolved and has now transitioned into molecular pathology routine laboratories. It is an attractive platform for analysing multiple genes at the same time with very little input material. Therefore, the need for high quality DNA obtained from automated DNA extraction systems has increased, especially to those laboratories which are dealing with formalin-fixed paraffin-embedded (FFPE) material and high sample throughput. This study evaluated five automated FFPE DNA extraction systems as well as five DNA quantification systems using the three most common techniques, UV spectrophotometry, fluorescent dye-based quantification and quantitative PCR, on 26 FFPE tissue samples. Additionally, the effects on downstream applications were analysed to find the most suitable pre-analytical methods for massively parallel sequencing in routine diagnostics. The results revealed that the Maxwell 16 from Promega (Mannheim, Germany) seems to be the superior system for DNA extraction from FFPE material. The extracts had a 1.3–24.6-fold higher DNA concentration in comparison to the other extraction systems, a higher quality and were most suitable for downstream applications. The comparison of the five quantification methods showed intermethod variations but all methods could be used to estimate the right amount for PCR amplification and for massively parallel sequencing. Interestingly, the best results in massively parallel sequencing were obtained with a DNA input of 15 ng determined by the NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). No difference could be detected in mutation analysis based on the results of the quantification methods. These findings emphasise, that it is particularly important to choose the most reliable and constant DNA extraction system, especially when using small biopsies and low elution volumes, and that all common DNA quantification techniques can be used for downstream applications like massively parallel sequencing.     

Single Nucleotide Polymorphism (SNP) Detection and Genotype Calling from Massively Parallel Sequencing (MPS) Data

Massively parallel sequencing (MPS), since its debut in 2005, has transformed the field of genomic studies. These new sequencing technologies have resulted in the successful identification of causal variants for several rare Mendelian disorders. They have also begun to deliver on their promise to explain some of the missing heritability from genome-wide association studies (GWAS) of complex traits. We anticipate a rapidly growing number of MPS-based studies for a diverse range of applications in the near future. One crucial and nearly inevitable step is to detect SNPs and call genotypes at the detected polymorphic sites from the sequencing data. Here, we review statistical methods that have been proposed in the past five years for this purpose. In addition, we discuss emerging issues and future directions related to SNP detection and genotype calling from MPS data.     

A beginners guide to SNP calling from high-throughput DNA-sequencing data

High-throughput DNA sequencing (HTS) is of increasing importance in the life sciences. One of its most prominent applications is the sequencing of whole genomes or targeted regions of the genome such as all exonic regions (i.e., the exome). Here, the objective is the identification of genetic variants such as single nucleotide polymorphisms (SNPs). The extraction of SNPs from the raw genetic sequences involves many processing steps and the application of a diverse set of tools. We review the essential building blocks for a pipeline that calls SNPs from raw HTS data. The pipeline includes quality control, mapping of short reads to the reference genome, visualization and post-processing of the alignment including base quality recalibration. The final steps of the pipeline include the SNP calling procedure along with filtering of SNP candidates. The steps of this pipeline are accompanied by an analysis of a publicly available whole-exome sequencing dataset. To this end, we employ several alignment programs and SNP calling routines for highlighting the fact that the choice of the tools significantly affects the final results.     

Whole-genome re-sequencing

DNA sequencing can be used to gain important information on genes, genetic variation and gene function for biological and medical studies. The growing collection of publicly available reference genome sequences will underpin a new era of whole genome re-sequencing, but sequencing costs need to fall and throughput needs to rise by several orders of magnitude. Novel technologies are being developed to meet this need by generating massive amounts of sequence that can be aligned to the reference sequence. The challenge is to maintain the high standards of accuracy and completeness that are hallmarks of the previous genome projects. One or more new sequencing technologies are expected to become the mainstay of future research, and to make DNA sequencing centre stage as a routine tool in genetic research in the coming years.