Evolution of genome sequencing techniques

The quality and the speed for genome sequencing has advanced at the same time that technology boundaries are stretched. This advancement has been divided so far in three generations. The first-generation methods enabled sequencing of clonal DNA populations. The second-generation massively increased throughput by parallelizing many reactions while the third-generation methods allow direct sequencing of single DNA molecules. The first techniques to sequence DNA were not developed until the mid-1970s, when two distinct sequencing methods were developed almost simultaneously, one by Alan Maxam and Walter Gilbert, and the other one by Frederick Sanger. The first one is a chemical method to cleave DNA at specific points and the second one uses ddNTPs, which synthesizes a copy from the DNA chain template. Nevertheless, both methods generate fragments of varying lengths that are further electrophoresed. Moreover, it is important to say that until the 1990s, the sequencing of DNA was relatively expensive and it was seen as a long process. Besides, using radiolabeled nucleotides also compounded the problem through safety concerns and prevented the automation. Some advancements within the first generation include the replacement of radioactive labels by fluorescent labeled ddNTPs and cycle sequencing with thermostable DNA polymerase, which allows automation and signal amplification, making the process cheaper, safer and faster. Another method is Pyrosequencing, which is based on the “sequencing by synthesis” principle. It differs from Sanger sequencing, in that it relies on the detection of pyrophosphate release on nucleotide incorporation. By the end of the last millennia, parallelization of this method started the Next Generation Sequencing (NGS) with 454 as the first of many methods that can process multiple samples, calling it the 2º generation sequencing. Here electrophoresis was completely eliminated. One of the methods that is sometimes used is SOLiD, based on sequencing by ligation of fluorescently dye-labeled di-base probes which competes to ligate to the sequencing primer. Specificity of the di-base probe is achieved by interrogating every 1st and 2nd base in each ligation reaction. The widely used Solexa/Illumina method uses modified dNTPs containing so called “reversible terminators” which blocks further polymerization. The terminator also contains a fluorescent label, which can be detected by a camera. Now, the previous step towards the third generation was in charge of Ion Torrent, who developed a technique that is based in a method of “sequencing-by-synthesis”. Its main feature is the detection of hydrogen ions that are released during base incorporation. Likewise, the third generation takes into account nanotechnology advancements for the processing of unique DNA molecules to a real time synthesis sequencing system like PacBio; and finally, the NANOPORE, projected since 1995, also uses Nano-sensors forming channels obtained from bacteria that conducts the sample to a sensor that allows the detection of each nucleotide residue in the DNA strand. The advancements in terms of technology that we have nowadays have been so quick, that it makes wonder: ¿How do we imagine the next generation

A new targeted CFTR mutation panel based on next-generation sequencing technology

Searching for mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR) is a key step in the diagnosis of and neonatal and carrier screening for cystic fibrosis (CF), and it has implications for prognosis and personalized therapy. The large number of mutations and genetic and phenotypic variability make this search a complex task. Herein, we developed, validated, and tested a laboratory assay for an extended search for mutations in CFTR using a next-generation sequencing based method, with a panel of 188 CFTR mutations customized for the Italian population. Overall, 1426 dried blood spots from neonatal screening, 402 genomic DNA samples from various origins, and 1138 genomic DNA samples from patients with CF were analyzed. The assay showed excellent analytical and diagnostic operative characteristics. We identified and experimentally validated 159 (of 188) CFTR mutations. The assay achieved detection rates of 95.0% and 95.6% in two large-scale case series of CF patients from central and northern Italy, respectively. These detection rates are among the highest reported so far with a genetic test for CF based on a mutation panel. This assay appears to be well suited for diagnostics, neonatal and carrier screening, and assisted reproduction, and it represents a considerable advantage in CF genetic counseling

Efficient depletion of host DNA contamination in malaria clinical sequencing.

The cost of whole-genome sequencing (WGS) is decreasing rapidly as next-generation sequencing technology continues to advance, and the prospect of making WGS available for public health applications is becoming a reality. So far, a number of studies have demonstrated the use of WGS as an epidemiological tool for typing and controlling outbreaks of microbial pathogens. Success of these applications is hugely dependent on efficient generation of clean genetic material that is free from host DNA contamination for rapid preparation of sequencing libraries. The presence of large amounts of host DNA severely affects the efficiency of characterizing pathogens using WGS and is therefore a serious impediment to clinical and epidemiological sequencing for health care and public health applications. We have developed a simple enzymatic treatment method that takes advantage of the methylation of human DNA to selectively deplete host contamination from clinical samples prior to sequencing. Using malaria clinical samples with over 80% human host DNA contamination, we show that the enzymatic treatment enriches Plasmodium falciparum DNA up to ∼9-fold and generates high-quality, nonbiased sequence reads covering >98% of 86,158 catalogued typeable single-nucleotide polymorphism loci

The Technology and Innovation Unit of the National Institute of Health: A sequencing and bioinformatics core facility specializing in public health genomics

The National Institute of Health (INSA) has a long tradition in investigating the molecular etiology of genetic and complex diseases. These activities greatly benefit from centralized sequencing services provided by the Technology and Innovation Unit (UTI). Its mission is to perform sequencing and genotyping assays in the framework of research, diagnosis and epidemiological surveillance, as well as to implement data analysis pipelines for the study of human gene variants. The equipment portfolio includes a NextSeq 550, a MiSeq, two 3500 AB Genetic Analyzers, a Fragment Analyzer and a 7500 Real-time PCR system. UTI provides results for average of 36.000 sequencing/fragment samples per year. The team has already performed >300 small genome, amplicon, gene panel (including clinical exome), 16S rRNA gene and RNA/microRNA next-generation sequencing assays for INSA and for several Universities in the scope of scientific collaborations. Technical procedures are conducted under a quality control system that includes external quality assessment for next-generation sequencing/Sanger sequencing and ISO 15189 accreditation for Sanger sequencing. UTI plays a key role in public health genomics, providing state-of-the-art equipment, centralized resources, technical expertise and short response times.This work was supported by Centre for Toxicogenomics and Human Health - UID/BIM/00009/2019 - and GenomePT project – POCI-01-0145-FEDER-022184info:eu-repo/semantics/publishedVersio

Virtual Environment for Next Generation Sequencing Analysis

Next Generation Sequencing technology, on the one hand, allows a more accurate analysis, and, on the other hand, increases the amount of data to process. A new protocol for sequencing the messenger RNA in a cell, known as RNA- Seq, generates millions of short sequence fragments in a single run. These fragments, or reads, can be used to measure levels of gene expression and to identify novel splice variants of genes. The proposed solution is a distributed architecture consisting of a Grid Environment and a Virtual Grid Environment, in order to reduce processing time by making the system scalable and flexibl

Clin Microbiol Infect

BackgroundWith the decreasing cost and efficiency of next generation sequencing, the technology is rapidly introduced into clinical and public health laboratory practice.AimsIn this review, the historical background and principles of first, second and third generation sequencing are described as are the characteristics of the most commonly used sequencing instruments.SourcesPeer reviewed literature, white papers and meeting reports.Content & implicationsNext generation sequencing is a technology that potentially could replace many traditional microbiological workflows, providing clinicians and public health specialists with more actionable information than hitherto achievable. Examples of the clinical and public health uses of the technology are provided. The challenge of comparability of different sequencing platforms is discussed. Finally, the future directions of the technology integrating it with laboratory management and public health surveillance systems, and moving it towards performing sequencing directly from the clinical specimen (metagenomics) could lead to yet another fundamental transformation of clinical diagnostics and public health surveillance.CC999999/Intramural CDC HHS/United States2018-06-01T00:00:00Z29074157PMC585721

Advancing transcriptome platforms

During the last decade of years, remarkable technological innovations have emerged that allow the direct or indirect determination of the transcriptome at unprecedented scale and speed. Studies using these methods have already altered our view of the extent and complexity of transcript profiling, which has advanced from one-gene-at-a-time to a holistic view of the genome. Here, we outline the major technical advances in transcriptome characterization, including the most popular used hybridization-based platform, the well accepted tag-based sequencing platform, and the recently developed RNA-Seq (RNA sequencing) based platform. Importantly, these next-generation technologies revolutionize assessing the entire transcriptome via the recent RNA-Seq technology

Next-Generation Sequencing in Equine Genomics

Next-generation sequencing of both DNA and RNA represents a second revolution in equine genetics following publication of the equine genome sequence. Technological advancements have resulted in a wide selection of next-generation sequencing platforms capable of completing small targeted experiments or resequencing complete genomes. DNA and RNA sequencing have applications in clinical and research environments. Standards for the validation and sharing of next-generation sequencing data are critical for the widespread application of the technology and applications discussed herein. As researchers and clinicians develop a better understanding of how genetic variation and phenotypic variation are linked, next-generation sequencing could help pave the way to personalized and precision management of horses