Long-Range PCR-Based NGS Applications to Diagnose Mendelian Retinal Diseases

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Abstract

The purpose of this study was to develop a flexible, cost-efficient, next-generation sequencing (NGS) protocol for genetic testing. Long-range polymerase chain reaction (PCR) amplicons of up to 20 kb in size were designed to amplify entire genomic regions for a panel ( n = 35) of inherited retinal disease (IRD)-associated loci. Amplicons were pooled and sequenced by NGS. The analysis was applied to 227 probands diagnosed with IRD: (A) 108 previously molecularly diagnosed, (B) 94 without previous genetic testing, and (C) 25 undiagnosed after whole-exome sequencing (WES). The method was validated with 100% sensitivity on cohort A. Long-range PCR-based sequencing revealed likely causative variant(s) in 51% and 24% of proband from cohorts B and C, respectively. Breakpoints of 3 copy number variants (CNVs) could be characterized. Long-range PCR libraries spike-in extended coverage of WES. Read phasing confirmed compound heterozygosity in 5 probands. The proposed sequencing protocol provided deep coverage of the entire gene, including intronic and promoter regions. Our method can be used (i) as a first-tier assay to reduce genetic testing costs, (ii) to elucidate missing heritability cases, (iii) to characterize breakpoints of CNVs at nucleotide resolution, (iv) to extend WES data to non-coding regions by spiking-in long-range PCR libraries, and (v) to help with phasing of candidate variants.

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Fast and accurate short read alignment with Burrows–Wheeler transform

Heng Li, Richard Durbin (2009)

Motivation: The enormous amount of short reads generated by the new DNA sequencing technologies call for the development of fast and accurate read alignment programs. A first generation of hash table-based methods has been developed, including MAQ, which is accurate, feature rich and fast enough to align short reads from a single individual. However, MAQ does not support gapped alignment for single-end reads, which makes it unsuitable for alignment of longer reads where indels may occur frequently. The speed of MAQ is also a concern when the alignment is scaled up to the resequencing of hundreds of individuals. Results: We implemented Burrows-Wheeler Alignment tool (BWA), a new read alignment package that is based on backward search with Burrows–Wheeler Transform (BWT), to efficiently align short sequencing reads against a large reference sequence such as the human genome, allowing mismatches and gaps. BWA supports both base space reads, e.g. from Illumina sequencing machines, and color space reads from AB SOLiD machines. Evaluations on both simulated and real data suggest that BWA is ∼10–20× faster than MAQ, while achieving similar accuracy. In addition, BWA outputs alignment in the new standard SAM (Sequence Alignment/Map) format. Variant calling and other downstream analyses after the alignment can be achieved with the open source SAMtools software package. Availability: http://maq.sourceforge.net Contact: rd@sanger.ac.uk

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SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing.

Anton Bankevich, Sergey Nurk, Dmitry Antipov … (2012)

The lion's share of bacteria in various environments cannot be cloned in the laboratory and thus cannot be sequenced using existing technologies. A major goal of single-cell genomics is to complement gene-centric metagenomic data with whole-genome assemblies of uncultivated organisms. Assembly of single-cell data is challenging because of highly non-uniform read coverage as well as elevated levels of sequencing errors and chimeric reads. We describe SPAdes, a new assembler for both single-cell and standard (multicell) assembly, and demonstrate that it improves on the recently released E+V-SC assembler (specialized for single-cell data) and on popular assemblers Velvet and SoapDeNovo (for multicell data). SPAdes generates single-cell assemblies, providing information about genomes of uncultivatable bacteria that vastly exceeds what may be obtained via traditional metagenomics studies. SPAdes is available online ( http://bioinf.spbau.ru/spades ). It is distributed as open source software.

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A framework for variation discovery and genotyping using next-generation DNA sequencing data

M.A. DePristo, E Banks, R.E. Poplin … (2011)

Recent advances in sequencing technology make it possible to comprehensively catalogue genetic variation in population samples, creating a foundation for understanding human disease, ancestry and evolution. The amounts of raw data produced are prodigious and many computational steps are required to translate this output into high-quality variant calls. We present a unified analytic framework to discover and genotype variation among multiple samples simultaneously that achieves sensitive and specific results across five sequencing technologies and three distinct, canonical experimental designs. Our process includes (1) initial read mapping; (2) local realignment around indels; (3) base quality score recalibration; (4) SNP discovery and genotyping to find all potential variants; and (5) machine learning to separate true segregating variation from machine artifacts common to next-generation sequencing technologies. We discuss the application of these tools, instantiated in the Genome Analysis Toolkit (GATK), to deep whole-genome, whole-exome capture, and multi-sample low-pass (~4×) 1000 Genomes Project datasets.

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Author and article information

Contributors

Tomasz Żarnowski: Role: Academic Editor

Journal

Journal ID (nlm-ta): Int J Mol Sci

Journal ID (iso-abbrev): Int J Mol Sci

Journal ID (publisher-id): ijms

Title: International Journal of Molecular Sciences

Publisher: MDPI

ISSN (Electronic): 1422-0067

Publication date (Electronic): 03 February 2021

Publication date Collection: February 2021

Volume: 22

Issue: 4

Electronic Location Identifier: 1508

Affiliations

[1 ]Institute of Medical Molecular Genetics, University of Zurich, 8952 Schlieren, Switzerland; maggi@ 123456medmolgen.uzh.ch (J.M.); koller@ 123456medmolgen.uzh.ch (S.K.); baehr@ 123456medmolgen.uzh.ch (L.B.); feil@ 123456medmolgen.uzh.ch (S.F.); kivrakpfiffner@ 123456medmolgen.uzh.ch (F.K.P.); maspoli@ 123456medmolgen.uzh.ch (A.M.)

[2 ]Department of Ophthalmology, University Hospital Zurich and University of Zurich, 8091 Zurich, Switzerland; james.hanson@ 123456usz.ch (J.V.M.H.); christina.gerth-kahlert@ 123456usz.ch (C.G.-K.)

[3 ]Zurich Center for Integrative Human Physiology (ZIHP), University of Zurich, 8057 Zurich, Switzerland

[4 ]Neuroscience Center Zurich (ZNZ), University and ETH Zurich, 8057 Zurich, Switzerland

Author notes

[* ]Correspondence: berger@ 123456medmolgen.uzh.ch ; Tel.: +41-44-556-33-50

Author information

Jordi Maggi https://orcid.org/0000-0002-9906-8739

Samuel Koller https://orcid.org/0000-0003-0965-0539

Christina Gerth-Kahlert https://orcid.org/0000-0001-6298-615X

Wolfgang Berger https://orcid.org/0000-0002-0370-3815

Article

Publisher ID: ijms-22-01508

DOI: 10.3390/ijms22041508

PMC ID: 7913364

PubMed ID: 33546218

SO-VID: 5feb6475-dae9-4b31-903f-5b27047b2abc

License:

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

Long-Range PCR-Based NGS Applications to Diagnose Mendelian Retinal Diseases

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Abstract

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Higher order chromatin architecture

Most cited references 109

Fast and accurate short read alignment with Burrows–Wheeler transform

SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing.

A framework for variation discovery and genotyping using next-generation DNA sequencing data

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