Technohub

Sequencing machines: Roche 454

The first NGS platform Talking about the system of the Roche 454 means starting from the dawn of Next Generation Sequencing. The first three Next Generation Sequencing (NGS) platforms were produced by three independent companies which, a little later, were acquired by other industrial groups. System 454 is based on pyrosequencing, a technique based on

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Low-pass genome sequencing: a new perspective for genome-wide CNV analysis

NGS, coverage depth, and CNV detection. Next-generation sequencing (NGS) has completely transformed the world of genetic testing, enabling us to sequence human whole exome or whole genome in one single experiment at an unprecedented scale, capacity and convenience. High-throughput sequencing based on short reads has empowered laboratories worldwide. We can now sift a huge amount

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Coverage in next-generation sequencing

What does «coverage» mean? Although the meaning of the term «coverage» may seem very simple, this term is often misused. In the context of Next-Generation Sequencing (NGS), coverage indicates the average number of reads that «cover» a specific target region. Coverage, therefore, always describes a relationship between the number of reads and a reference region

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What’s the right price for whole exome sequencing?

What’s the right price of whole exome sequencing? This is one of the most frequent questions I am being asked and one of the most searched on the internet: what’s the price of whole exome sequencing (which consists of the sequencing of all human genes)? Of note, whole exome sequencing does not consist in the

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Turnaround time in genetic testing for rare disorders

Genetic testing: a complex workflow. Patients and physicians sometimes complain about the turnaround time (TAT) of genetic testing. Now, it should be mentioned that genetic testing is technically complex, requiring several steps of sample manipulation. Especially for what concerns genetic testing for rare disorders, there’s also a final step, maybe the most important one, which

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Bed files in Next Generation Sequencing

What are the bed files? If you are interested, or directly involved, in Next Generation Sequencing applications for research or clinical diagnostics, especially in whole exome sequencing or targeted multigene panel testing, you’ve certainly heard of the so-called bed files. If you haven’t heard of them yet, you’ll soon need to learn what they are

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Amino acid codes (symbols)

Amino acids codes (symbols) are listed below (1 and 3 letters codes). Approved nomenclature for reports is the 3 letters code: Amino acid Code (1 letter) Code (3 letters) (official) Alanine A Ala Arginine R Arg Asparagine N Asn Aspartic Acid D Asp Cysteine C Cys Glutamic Acid E Glu Glutamine Q Gln Glicine G

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Sequencing the DNA of a single cell

Overview Whole genome sequencing on DNA of a single cell has been reported by several authors. Singe cell genome sequencing is of interest in a number of niche applications, such as the measuring of the genomic diversity in one individual’s gamete genomes, the mutation phase determination, and somatic DNA changes in tumors or after exposure to mutagenic agents.

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Sequencing library: what is it?

Summary The sequencing library’s preparation is the first step in any Next Generation Sequencing analysis. There are different ways to prepare a sequencing library, depending on the sequencing platform (Life Technologies, Illumina, Roche, Pacific Biosciences) and the planned analysis (whole genome sequencing, whole exome sequencing, targeted DNA sequencing, whole-transcriptome sequencing, targeted RNA sequencing, ChIP-seq, RIP-seq, epigenetic

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RNA extraction tips

A vulnerable molecule RNA extraction from biological samples is complicated by the vulnerability of the RNA molecule itself and by the ubiquitous presence of ribonuclease enzymes in cells, tissues, and environment which can rapidly degrade RNA (for instance, RNase 7, a member of the RNase A superfamily is secreted by human skin and serves as

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GATK

GATK: the Genome Analysis Toolkit The Genome Analysis Toolkit or more simply, GATK, is a software which is widely used to analyze high throughput sequencing data. GATK has been developed by the Data Science and Data Engineering group working at the Broad Institute. This specialized toolkit provides users with a broad selection tools and to focus on variant discovery, genotyping and data quality

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NGS for dummies

Few simple words to explain NGS After years of Sanger sequencing being adopted as the gold standard in molecular diagnostics, Next Generation Sequencing (NGS) is about to finally an completely take over. NGS is also known as high-throughput sequencing (high yield sequencing) as it allows to sequence many fragments in parallel (which is impossible by traditional Sanger sequencing). There

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CNVs: detection methods

WHOLE & NOT Molecular diagnostics at present can be subdivided in «whole» approach (Whole Exome or Whole Genome) and «targeted» approach (single gene or single panel testing). Methods for the detection of CNVs vary significantly on the desired type of approach. Here below a guide to the right choice (and the techniques we have chosen). «WHOLE»

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CNVs databases

STORING IMBALANCES Due to the amount and complexity of information relating to Copy Number Variations (CNVs), the scientific community felt the need to organize all available data, and future submissions, into open-source online repositories. Luckily, some of these resources are reciprocally synchronized, so that conflicting information is reduced to the minimum. The most important CNVs

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ClinVar or HGMD?

Which one is the best database? Genetic testing remains the most complex and difficult among all laboratory tests, both from the technical and the medical point of view. Since an excellent bioinformatic pipeline is fundamental to produce good data for the geneticist and because several bioinformatic tools are available today, it is necessary to choose well when establishing standard

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MLPA: 10 questions and answers

MLPA: Q&A MLPA is acronym for Multiplex Ligation Probe Amplification, which is a special molecular technique based on PCR. It is used to scan large deletions/duplications/multiplications which are not detectable by standard sequencing (either NGS or Sanger). The alternative to this technique is the qPCR (quantitative PCR). Is it necessary to always perform MLPA? No, it isn’t always indicated. Multiplex

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Polyphen/Polyphen-2

A tool to predict the effect of missense variants Polyphen, now available in its version Polyphen-2, predicts the impact of a missense variant (also referred to as nsSNP: non synonymous single nucleotide polymorphism) based on (1) protein sequence (2) phylogenetic information and (3) structural information. So Polyphen is able to do the so called functional annotation of missense variants.

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dbVar: understanding structural variations

Besides DGV, DGVa and DECIPHER, there is another database that lists structural DNA variations: it’s dbVar, which we expect to be the most comprehensive one, based on its illustrious trademark: NCBI. What is a structural variation? Before going on, we just want to recall here that a genomic structural variation or variant is usually defined

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