Coverage (genetics)

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An overlap of the product of three sequencing runs, with the read sequence coverage at each point indicated. Read, read length and read depth to achieve a read depth of 4.jpg
An overlap of the product of three sequencing runs, with the read sequence coverage at each point indicated.

In genetics, coverage is one of several measures of the depth or completeness of DNA sequencing, and is more specifically expressed in any of the following terms:

Contents

Sequence coverage

Rationale

Even though the sequencing accuracy for each individual nucleotide is very high, the very large number of nucleotides in the genome means that if an individual genome is only sequenced once, there will be a significant number of sequencing errors. Furthermore, many positions in a genome contain rare single-nucleotide polymorphisms (SNPs). Hence to distinguish between sequencing errors and true SNPs, it is necessary to increase the sequencing accuracy even further by sequencing individual genomes a large number of times.

Ultra-deep sequencing

The term "ultra-deep" can sometimes also refer to higher coverage (>100-fold), which allows for detection of sequence variants in mixed populations. [5] [6] [7] In the extreme, error-corrected sequencing approaches such as Maximum-Depth Sequencing can make it so that coverage of a given region approaches the throughput of a sequencing machine, allowing coverages of >10^8. [8]

Transcriptome sequencing

Deep sequencing of transcriptomes, also known as RNA-Seq, provides both the sequence and frequency of RNA molecules that are present at any particular time in a specific cell type, tissue or organ. [9] Counting the number of mRNAs that are encoded by individual genes provides an indicator of protein-coding potential, a major contributor to phenotype. [10] Improving methods for RNA sequencing is an active area of research both in terms of experimental and computational methods. [11]

Calculation

The average coverage for a whole genome can be calculated from the length of the original genome (G), the number of reads (N), and the average read length (L) as . For example, a hypothetical genome with 2,000 base pairs reconstructed from 8 reads with an average length of 500 nucleotides will have 2× redundancy. This parameter also enables one to estimate other quantities, such as the percentage of the genome covered by reads (sometimes also called breadth of coverage). A high coverage in shotgun sequencing is desired because it can overcome errors in base calling and assembly. The subject of DNA sequencing theory addresses the relationships of such quantities. [2]

Physical coverage

Sometimes a distinction is made between sequence coverage and physical coverage. Where sequence coverage is the average number of times a base is read, physical coverage is the average number of times a base is read or spanned by mate paired reads. [2] [12] [4]

Genomic coverage

In terms of genomic coverage and accuracy, whole genome sequencing can broadly be classified into either of the following: [13]

Producing a truly high-quality finished sequence by this definition is very expensive. Thus, most human "whole genome sequencing" results are draft sequences (sometimes above and sometimes below the accuracy defined above). [13]

Related Research Articles

In genetics, shotgun sequencing is a method used for sequencing random DNA strands. It is named by analogy with the rapidly expanding, quasi-random shot grouping of a shotgun.

<span class="mw-page-title-main">Human genome</span> Complete set of nucleic acid sequences for humans

The human genome is a complete set of nucleic acid sequences for humans, encoded as DNA within the 23 chromosome pairs in cell nuclei and in a small DNA molecule found within individual mitochondria. These are usually treated separately as the nuclear genome and the mitochondrial genome. Human genomes include both protein-coding DNA sequences and various types of DNA that does not encode proteins. The latter is a diverse category that includes DNA coding for non-translated RNA, such as that for ribosomal RNA, transfer RNA, ribozymes, small nuclear RNAs, and several types of regulatory RNAs. It also includes promoters and their associated gene-regulatory elements, DNA playing structural and replicatory roles, such as scaffolding regions, telomeres, centromeres, and origins of replication, plus large numbers of transposable elements, inserted viral DNA, non-functional pseudogenes and simple, highly-repetitive sequences. Introns make up a large percentage of non-coding DNA. Some of this non-coding DNA is non-functional junk DNA, such as pseudogenes, but there is no firm consensus on the total amount of junk DNA.

<span class="mw-page-title-main">Genomics</span> Discipline in genetics

Genomics is an interdisciplinary field of biology focusing on the structure, function, evolution, mapping, and editing of genomes. A genome is an organism's complete set of DNA, including all of its genes as well as its hierarchical, three-dimensional structural configuration. In contrast to genetics, which refers to the study of individual genes and their roles in inheritance, genomics aims at the collective characterization and quantification of all of an organism's genes, their interrelations and influence on the organism. Genes may direct the production of proteins with the assistance of enzymes and messenger molecules. In turn, proteins make up body structures such as organs and tissues as well as control chemical reactions and carry signals between cells. Genomics also involves the sequencing and analysis of genomes through uses of high throughput DNA sequencing and bioinformatics to assemble and analyze the function and structure of entire genomes. Advances in genomics have triggered a revolution in discovery-based research and systems biology to facilitate understanding of even the most complex biological systems such as the brain.

<span class="mw-page-title-main">DNA sequencer</span> A scientific instrument used to automate the DNA sequencing process

A DNA sequencer is a scientific instrument used to automate the DNA sequencing process. Given a sample of DNA, a DNA sequencer is used to determine the order of the four bases: G (guanine), C (cytosine), A (adenine) and T (thymine). This is then reported as a text string, called a read. Some DNA sequencers can be also considered optical instruments as they analyze light signals originating from fluorochromes attached to nucleotides.

In bioinformatics, sequence assembly refers to aligning and merging fragments from a longer DNA sequence in order to reconstruct the original sequence. This is needed as DNA sequencing technology might not be able to 'read' whole genomes in one go, but rather reads small pieces of between 20 and 30,000 bases, depending on the technology used. Typically, the short fragments (reads) result from shotgun sequencing genomic DNA, or gene transcript (ESTs).

<span class="mw-page-title-main">DNA sequencing</span> Process of determining the nucleic acid sequence

DNA sequencing is the process of determining the nucleic acid sequence – the order of nucleotides in DNA. It includes any method or technology that is used to determine the order of the four bases: adenine, guanine, cytosine, and thymine. The advent of rapid DNA sequencing methods has greatly accelerated biological and medical research and discovery.

<span class="mw-page-title-main">ABI Solid Sequencing</span>

SOLiD (Sequencing by Oligonucleotide Ligation and Detection) is a next-generation DNA sequencing technology developed by Life Technologies and has been commercially available since 2006. This next generation technology generates 108 - 109 small sequence reads at one time. It uses 2 base encoding to decode the raw data generated by the sequencing platform into sequence data.

<span class="mw-page-title-main">Whole genome sequencing</span> Determining nearly the entirety of the DNA sequence of an organisms genome at a single time.

Whole genome sequencing (WGS), also known as full genome sequencing, complete genome sequencing, or entire genome sequencing, is the process of determining the entirety, or nearly the entirety, of the DNA sequence of an organism's genome at a single time. This entails sequencing all of an organism's chromosomal DNA as well as DNA contained in the mitochondria and, for plants, in the chloroplast.

<span class="mw-page-title-main">RNA-Seq</span> Lab technique in cellular biology

RNA-Seq is a sequencing technique which uses next-generation sequencing (NGS) to reveal the presence and quantity of RNA in a biological sample at a given moment, analyzing the continuously changing cellular transcriptome.

Cap analysis gene expression (CAGE) is a gene expression technique used in molecular biology to produce a snapshot of the 5′ end of the messenger RNA population in a biological sample. The small fragments from the very beginnings of mRNAs are extracted, reverse-transcribed to cDNA, PCR amplified and sequenced. CAGE was first published by Hayashizaki, Carninci and co-workers in 2003. CAGE has been extensively used within the FANTOM research projects.

SOAP is a suite of bioinformatics software tools from the BGI Bioinformatics department enabling the assembly, alignment, and analysis of next generation DNA sequencing data. It is particularly suited to short read sequencing data.

De novo transcriptome assembly is the de novo sequence assembly method of creating a transcriptome without the aid of a reference genome.

<span class="mw-page-title-main">Illumina dye sequencing</span>

Illumina dye sequencing is a technique used to determine the series of base pairs in DNA, also known as DNA sequencing. The reversible terminated chemistry concept was invented by Bruno Canard and Simon Sarfati at the Pasteur Institute in Paris. It was developed by Shankar Balasubramanian and David Klenerman of Cambridge University, who subsequently founded Solexa, a company later acquired by Illumina. This sequencing method is based on reversible dye-terminators that enable the identification of single nucleotides as they are washed over DNA strands. It can also be used for whole-genome and region sequencing, transcriptome analysis, metagenomics, small RNA discovery, methylation profiling, and genome-wide protein-nucleic acid interaction analysis.

In DNA sequencing, a read is an inferred sequence of base pairs corresponding to all or part of a single DNA fragment. A typical sequencing experiment involves fragmentation of the genome into millions of molecules, which are size-selected and ligated to adapters. The set of fragments is referred to as a sequencing library, which is sequenced to produce a set of reads.

G&T-seq is a novel form of single cell sequencing technique allowing one to simultaneously obtain both transcriptomic and genomic data from single cells, allowing for direct comparison of gene expression data to its corresponding genomic data in the same cell...

<span class="mw-page-title-main">Duplex sequencing</span>

Duplex sequencing is a library preparation and analysis method for next-generation sequencing (NGS) platforms that employs random tagging of double-stranded DNA to detect mutations with higher accuracy and lower error rates.

Third-generation sequencing is a class of DNA sequencing methods currently under active development.

Transcriptomics technologies are the techniques used to study an organism's transcriptome, the sum of all of its RNA transcripts. The information content of an organism is recorded in the DNA of its genome and expressed through transcription. Here, mRNA serves as a transient intermediary molecule in the information network, whilst non-coding RNAs perform additional diverse functions. A transcriptome captures a snapshot in time of the total transcripts present in a cell. Transcriptomics technologies provide a broad account of which cellular processes are active and which are dormant. A major challenge in molecular biology is to understand how a single genome gives rise to a variety of cells. Another is how gene expression is regulated.

A plant genome assembly represents the complete genomic sequence of a plant species, which is assembled into chromosomes and other organelles by using DNA fragments that are obtained from different types of sequencing technology.

References

  1. ↑ "Sequencing Coverage". illumina.com. Illumina education. Retrieved 2020-10-08.
  2. 1 2 3 Sims, David; Sudbery, Ian; Ilott, Nicholas E.; Heger, Andreas; Ponting, Chris P. (2014). "Sequencing depth and coverage: key considerations in genomic analyses". Nature Reviews Genetics. 15 (2): 121–132. doi:10.1038/nrg3642. PMID   24434847. S2CID   13325739.
  3. ↑ Mardis, Elaine R. (2008-09-01). "Next-Generation DNA Sequencing Methods". Annual Review of Genomics and Human Genetics. 9 (1): 387–402. doi:10.1146/annurev.genom.9.081307.164359. ISSN   1527-8204. PMID   18576944.
  4. 1 2 Ekblom, Robert; Wolf, Jochen B. W. (2014). "A field guide to whole‐genome sequencing, assembly and annotation". Evolutionary Applications. 7 (9): 1026–42. doi:10.1111/eva.12178. PMC   4231593 . PMID   25553065.
  5. ↑ Ajay SS, Parker SC, Abaan HO, Fajardo KV, Margulies EH (September 2011). "Accurate and comprehensive sequencing of personal genomes". Genome Res. 21 (9): 1498–505. doi:10.1101/gr.123638.111. PMC   3166834 . PMID   21771779.
  6. ↑ Mirebrahim, Hamid; Close, Timothy J.; Lonardi, Stefano (2015-06-15). "De novo meta-assembly of ultra-deep sequencing data". Bioinformatics. 31 (12): i9–i16. doi:10.1093/bioinformatics/btv226. ISSN   1367-4803. PMC   4765875 . PMID   26072514.
  7. ↑ Beerenwinkel, Niko; Zagordi, Osvaldo (2011-11-01). "Ultra-deep sequencing for the analysis of viral populations". Current Opinion in Virology. 1 (5): 413–418. doi:10.1016/j.coviro.2011.07.008. PMID   22440844.
  8. ↑ Jee, J.; Rasouly, A.; Shamovsky, I.; Akivis, Y.; Steinman, S.; Mishra, B.; Nudler, E. (2016). "Rates and mechanisms of bacterial mutagenesis from maximum-depth sequencing". Nature. 534 (7609): 693–696. Bibcode:2016Natur.534..693J. doi:10.1038/nature18313. PMC   4940094 . PMID   27338792.
  9. ↑ Malone, John H.; Oliver, Brian (2011-01-01). "Microarrays, deep sequencing and the true measure of the transcriptome". BMC Biology. 9: 34. doi:10.1186/1741-7007-9-34. ISSN   1741-7007. PMC   3104486 . PMID   21627854.
  10. ↑ Hampton M, Melvin RG, Kendall AH, Kirkpatrick BR, Peterson N, Andrews MT (2011). "Deep sequencing the transcriptome reveals seasonal adaptive mechanisms in a hibernating mammal". PLOS ONE. 6 (10): e27021. Bibcode:2011PLoSO...627021H. doi: 10.1371/journal.pone.0027021 . PMC   3203946 . PMID   22046435.
  11. ↑ Heyer EE, Ozadam H, Ricci EP, Cenik C, Moore MJ (2015). "An optimized kit-free method for making strand-specific deep sequencing libraries from RNA fragments". Nucleic Acids Res. 43 (1): e2. doi:10.1093/nar/gku1235. PMC   4288154 . PMID   25505164.
  12. ↑ Meyerson, M.; Gabriel, S.; Getz, G. (2010). "Advances in understanding cancer genomes through second-generation sequencing". Nature Reviews Genetics. 11 (10): 685–696. doi:10.1038/nrg2841. PMID   20847746. S2CID   2544266.
  13. 1 2 Kris A. Wetterstrand, M.S. "The Cost of Sequencing a Human Genome". National Human Genome Research Institute . Last updated: November 1, 2021
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