how are genome sequences assembled

The genome

sequence is then assembled by aligning sequences of adjacent clones and calculating a path through these alignments that will produce a non-redundant sequence. Typically, evaluations of these alignments are guided by a map (often called a Tiling Path (or TPF)).

Full Answer

What are the different methods of genome sequence assembly?

A genome sequence assembly can be performed in two ways: mapping and assembly, or de novo assembly.

What is a genome assembly?

Genome assembly refers to the process of putting nucleotide sequence into the correct order. Assembly is required, because sequence read lengths – at least for now – are much shorter than most genomes or even most genes.

What is sequence assembly?

Sequence assembly. 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 cannot read whole genomes in one go, but rather reads small pieces of between 20 and 30000 bases,...

How many reads does it take to sequence a full genome?

Thus, a genome must be fragmented, sequenced in bits and then re-assembled to obtain the full contiguous sequence. Each sequenced piece of DNA is referred to as a sequencing read (read for short). Several thousand to several million reads must be produced to reconstruct the sequence of a longer molecule.

How is a genome assembled?

To assemble a genome, computer programs typically use data consisting of single and paired reads. Single reads are simply the short sequenced fragments themselves; they can be joined up through overlapping regions into a continuous sequence known as a 'contig'.

How is sequence assembly done?

Sequence assembly can be done using one of three approaches: (1) greedy, (2) overlap-layout-consensus (OLC) and Hamiltonian path, and (3) de Bruijn graph and Eulerian pathd.

What are the two ways to assemble a genetic sequence?

There are two broad types of assembly techniques that may be utilized: de novo and comparative assembly. De novo assembly is used for new genomes that have not been previously sequenced or are not similar to genomes that have previously been sequenced.

Why is it difficult to assemble a genome?

4.1. Repeat sequences are difficult to assemble since high-identity reads could come from different portions of the genome, generating gaps, ambiguities and collapses in alignment and assembly, which, in turn, can produce biases and errors when interpreting results.

What is a complete genome assembly?

Genome assembly is the computational process of deciphering the sequence composition of the genetic material (DNA) within the cell of an organism, using numerous short sequences called reads derived from different portions of the target DNA as input.

What do you mean by sequence assembly?

In bioinformatics, sequence assembly refers to aligning and merging fragments from a longer DNA sequence in order to reconstruct the original sequence.

What makes a good genome assembly?

A high quality genome assembly is expected to contain a higher number of complete and single copy BUSCO genes (C&S) and a lower number of missing (M) or fragmented (F) BUSCO genes [8].

What is the first step of genome sequencing?

The first phase, called the shotgun phase, divided human chromosomes into DNA segments of an appropriate size, which were then further subdivided into smaller, overlapping DNA fragments that were sequenced.

Is it possible to assemble a genome without aligning it to a reference genome?

There are two different types of genome assembly: de novo assembly and mapping to a reference genome (also known as reference-based alignment). De novo assembly refers to the genome assembly of a novel genome from scratch without the aid of reference genomic data.

What is genome sequence assembly software?

ARACHNE is a program for assembling data from whole genome shotgun sequencing experiments. It was designed for long reads from Sanger sequencing technology, and has been used extensively to assemble many genomes, including many that are large and highly repetitive. Reconciliator 2.0 – The tool for Merging Assemblies.

How does Illumina sequencing work?

Illumina sequencing technology leverages clonal array formation and proprietary reversible terminator technology for rapid and accurate large-scale sequencing. The innovative and flexible sequencing system enables a broad array of applications in genomics, transcriptomics, and epigenomics.

What is assembly algorithm?

Genome assembly algorithms are sets of well defined procedures for reconstructing DNA sequences from large numbers of shorter DNA sequence fragments. Fragments are aligned against one another and overlapping sections are identified and merged.

How does Illumina sequencing work?

Illumina sequencing technology leverages clonal array formation and proprietary reversible terminator technology for rapid and accurate large-scale sequencing. The innovative and flexible sequencing system enables a broad array of applications in genomics, transcriptomics, and epigenomics.

What is genome sequence assembly software?

ARACHNE is a program for assembling data from whole genome shotgun sequencing experiments. It was designed for long reads from Sanger sequencing technology, and has been used extensively to assemble many genomes, including many that are large and highly repetitive. Reconciliator 2.0 – The tool for Merging Assemblies.

How does shotgun sequencing work?

​Shotgun Sequencing The method involves randomly breaking up the genome into small DNA fragments that are sequenced individually. A computer program looks for overlaps in the DNA sequences, using them to reassemble the fragments in their correct order to reconstitute the genome.

How does de novo assembly work?

De novo sequencing refers to sequencing a novel genome where there is no reference sequence available for alignment. Sequence reads are assembled as contigs, and the coverage quality of de novo sequence data depends on the size and continuity of the contigs (ie, the number of gaps in the data).

How is genome sequence assembly performed?

A genome sequence assembly can be performed in two ways: mapping and assembly, or de novo assembly. If the genome has been sequenced before and a reference genome sequence already exists, then the newly obtained resequence reads are first mapped to the reference genome through alignment and then assembled in proper order; this mode of assembly is called “mapping and assembly.” Bowtie is an ultrafast, memory-efficient short-read aligner that helps in mapping and assembly . It rapidly aligns large sets of short sequencing reads to a reference sequence, at a rate of over 25 million 35-bp reads per hour. For reads longer than about 50 bp, Bowtie 2 is generally faster, more sensitive, and uses less memory than the original Bowtie ( http://bowtie-bio.sourceforge.net/index.shtml ).

How does genome assembly work?

Therefore, genome assembly is a hierarchical process; it is performed in steps beginning from the assembly of the sequence reads into contigs, assembly of the contigs into scaffolds (supercontigs), and assembly of the scaffolds into chromosomes. Many genome assemblies remain restricted to scaffold level for a long time because the gaps can not be easily sequenced. Some scaffolds can be placed within a chromosome, while the chromosomal assignment of other scaffolds may remain difficult.

How to evaluate de novo genome assembly?

One widely used metric to evaluate the quality of assembly is the contig and scaffold N50 value (see Box 7.1 ). An N50 contig is the size of the shortest contig such that the sum of contigs of that size or longer constitutes at least 50% of the total size of the assembled contigs. For example, an N50 contig of 100 kb means that when contigs of 100 kb or longer are added up, the resulting size represents at least 50% of the total size of all assembled contigs. Likewise, an N50 scaffold size is the length of the shortest scaffold such that the sum of the scaffolds of that size or longer constitutes at least 50% of the total size of all assembled scaffolds.

How many insect genomes are there?

There are 31 insect genome assemblies available at the National Center for Biotechnology Information (NCBI) Genome Project Database (ncbi.nlm.nih.gov/entrez): 19 genome assemblies are available from Dipteran species, including 1 Hessian fly ( http://www.ncbi.nlm.nih.gov/genomeprj/45867) and 6 mosquitoes ( Holt et al., 2002; Nene et al., 2007; Arensburger et al., 2010; Lawniczak et al., 2010; http://www.ncbi.nlm.nih.gov/genomeprj/46227 ); and 12 Drosophila ( Drosophila 12 Genomes Consortium, 2007 ). There are assemblies from seven Hymenopteran species, including the honeybee ( Honeybee Genome Sequencing Consortium, 2006 ), three ants ( Bonasio et al., 2010; http://www.ncbi.nlm.nih.gov/genomeprj/48091 ), and three wasps ( Werren et al., 2010 ). There are also assemblies from one Lepidopteran species, Bombyx mori ( Xia et al., 2004; Mita et al., 2004 ), and one Coleopteran species, Tribolium castaneum ( Tribolium Genome Sequencing Consortium, 2008 ). Assemblies from three hemimetabolous insects are available, including one Phthiraptern insect body louse ( Kirkness et al., 2010) and two Hemipteran ( The International Aphid Genomics Consortium, 2010; http://www.ncbi.nlm.nih.gov/genomeprj/13648 ). In addition, a number of insect genomes are being sequenced using “next-generation” approaches, and it is anticipated that rapid expansion of sequenced genomes will bring tremendous opportunities to the investigation of TE diversity and evolution. Whole-genome comparative analysis of insect TEs is still in its early stages, and a few interesting observations are highlighted below. Systematic analysis of the 12 Drosophila genomes revealed that while the TE content varies from 2.7% to ~25% of the host genomes, the relative abundance of different groups of TEs is conserved across most of the species ( Drosophila 12 Genomes Consortium, 2007 ). Comprehensive analysis identified over 100 potential horizontal transfer events by more than 20 TEs among the 12 Drosophila species, most of which involved DNA transposons and LTR retrotransposons ( Loreto et al., 2008; Bartolome et al., 2009 ). Systematic comparison of multiple aligned genomes revealed TE insertion sites across the entire genomes, and supported a hypothesis that most TEs in D. melanogaster are recently active ( Caspi and Pachter, 2006 ). The published genomes of Anopheles, Culex, and Aedes mosquitoes vary by five-fold in size, ranging from ~270 Mbp for An. gambiae ( Holt et al., 2002) to ~500 Mbp for C. quinquefasciatus ( Arensburger et al., 2010 ), and ~1300 Mbp for Ae. aegypti ( Nene et al., 2007 ). TE contents in these three species are 11–16%, 29%, and 47% of the assembled genomes, respectively, indicating that TEs contributed significantly to the genome size variations among mosquito species. While 16% of the Ae. aegypti genome is occupied by MITE-like elements, cut-and-paste DNA transposons represent only 3% of the genome, suggesting that a small number of DNA transposons may be responsible for cross-mobilizing a large number of non-autonomous MITE-like sequences ( Nene et al., 2007 ). Systematic comparisons also revealed an apparent horizontal transfer event between Aedes and Anopheles mosquitoes involving an ITmD37E DNA transposon ( Biedler and Tu, 2007 ). Among the sequenced Hymenopteran species, the honeybee genome contains only ~7% repetitive sequences while repeat contents range from 15 to 27% in the ants and wasps ( Honeybee Genome Sequencing Consortium, 2006; Bonasio et al., 2010; Werren et al., 2010 ). The parasitic body louse harbors only a very small number of TEs, which occupy 1% of its 110-Mbp genome ( Kirkness et al., 2010 ).

What is genome assembly?

Genom e assemblies represent models for the actual genome—and thus are never perfect. A single assembly cannot represent all the diversity within populations of a species, and it is nearly impossible to eliminate all possible technological or algorithmical errors. Therefore, published genomes that have an active research community are continuously improved. For instance, in Dec. 2013, a new version of the human genome assembly was released (build 38), with several improvements compared to build 37, first released in 2009.

What is the process of putting a large number of short DNA sequences back together?

Genome assembly refers to the process of taking a large number of short DNA sequences and putting them back together to create a representation of the original chromosomes from which the DNA originated.

Why is genome assembly important?

Genome assembly refers to the process of putting nucleotide sequence into the correct order. Assembly is required, because sequence read lengths – at least for now – are much shorter than most genomes or even most genes. Genome assembly is made easier by the existence of public databases, freely available on the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov ). Just as it is much easier to assemble a picture puzzle if you know what the picture looks like, it is much easier to assemble genes and genomes if you have a good idea of the sequence order. In the human genome, genes occur in the same physical location on the chromosome, but there can be different numbers of copies and variable numbers of repeated sequence that complicate assembly. Although bacterial genomes are much smaller, genes are not necessarily in the same location and multiple copies of the same gene may appear in different locations on the genome. Therefore even with the availability of commercial software and ever growing reference databases, the process of genome assembly can take considerably longer than the time to obtain actual sequence.

How is genome sequence accomplished?

To accomplish genome sequencing, the genome is fragmented and small pieces are sequenced many times. These sequences are then assembled to try to recreate the chromosome sequences (see Assembly Basics for more information).

How to access genome sequences?

Genome sequences and annotation can be accessed by following the links to the FTP site from the page for the assembly of interest in the NCBI Assembly resource or by navigating the NCBI genomes FTP site. See the Genomes Download FAQ for more details.

What is the golden path in genome sequencing?

When genome sequencing initially started it was thought that the genome assembly could be represented by a single 'Golden Path'. That is, a single set of overlapping sequences could be selected to produce a non-redundant chromosome sequence (with gaps) that would fully represent the sequence at all loci. It was thought that the predominant form of variation was single nucleotide polymorphism ( SNP) and these polymorphisms would be represented as annotation on the chromosome sequence. Subsequent genome analysis has shown that this model will not work for some parts of the genome. Large-scale structural variations, often in the form of Copy Number Variation (CNVs) are more prevalent than originally thought (see dbVar for more information). If a genome contains regions with complex allelic diversity, it may be necessary to produce more than 1 sequence path to fully represent that region. For example, the current public human reference assembly ( GRCh38) has 8 different paths through the MHC region, a region known to have a high degree of allelic complexity. To accommodate this increased complexity, we have developed a more robust data model. Terms used in this model are defined below and a graphical representation is shown in figure 2.

How many times are chromosomes represented?

Any locus may be represented 0 or 1 time, and entire chromosomes are only represented 0 or 1 times .

What is the term for the set of chromosomes, unlocalized and unplaced, and alternate?

Assembly : The set of chromosomes, unlocalized and unplaced (sometimes called 'random') and alternate sequences used to represent an organism's genome. Assemblies are constructed from 1 or more assembly units.

What are genomic regions?

Genomic regions are defined on the Primary Assembly. These regions point to scaffolds contained within the alternate locus units. Note: not all alternate locus scaffolds will be associated with a region.

Where are regions located?

Regions are locations on the primary assembly (typically on the chromosome sequences) for which alternate representations or genome patches exist.

What is the process of assembling a genome?

This process is called assembly. Assembly is like solving a jigsaw puzzle. Special software tools called assemblers are used to assemble these reads according to how they overlap, in order to generate continuous strings called contigs. These contigs can be the whole genome itself, or parts of the genome (as shown in Figure 2).

What is the genome?

A genome is considered as all the genetic material, including all the genes of an organism. The genome contains all the information of an organism that is required to build and maintain it.

What can go wrong in Genome Assembly?

Genomes contain patterns of nucleic acids that occur many times across the genome. These structures are called repeats. These repeats can complicate the assembly process and result in ambiguities.

What is the second type of assembler?

The second type of assembler is the de Bruijn graph (DBG) method [2]. Rather than using the complete reads as they are, the DBG method breaks reads into shorter fragments called k -mers (with length k) and then build a de Bruijn graph using all the k -mers. Finally, the genome sequences are inferred based on the de Bruijn graph. SPAdes is a popular assembler which is based on the DBG method.

What is PacBio sequencing?

PacBio is a third-generation sequencing technology which produces long reads. Image by KENNETH RODRIGUES from Pixabay (CC0) Special machines, known as sequencing machines are used to extract short random sequences from the genome we are interested in. Current sequencing technologies cannot read the whole genome at once.

How many base pairs does NODE_1 have?

From the Icarus contig browser, we can see that the contig named NODE_1 maps very closely to the reference genome of COVID-19. It has a genome fraction of 99.99% (as shown in Figure 3). Moreover, the total aligned length of 29,900 base pairs is very close to the length of the reference genome which is 29,903 base pairs.

Can a sequencing machine read the entire genome?

We cannot guarantee that the sequencing machine can produce reads covering the entire genome. The sequencing machine may miss some parts of the genome and there won’t be reads covering that region. This will affect the assembly process and those missed regions will not be present in the final assembly.

How is a genome sequence assembled?

The genome sequence is then assembled by aligning sequences of adjacent clones and calculating a path through these alignments that will produce a non-redundant sequence. Typically, evaluations of these alignments are guided by a map (often called a Tiling Path (or TPF) ). Examples of such programs are Gigassembler (Jim Kent, UCSC) and TPF Analyzer (Richa Agarwala, NCBI). It should be noted that the clone sequence need not be finished in order to produce a genome assembly. Indeed, the first several human assemblies consisted of a mixture of finished and unfinished sequence. Typically, unfinished sequence is deposited to the High Throughput Genome Sequences (HTGS ) division of GenBank. Once it is finished it is moved to the regular divisions. In order to assess unfinished sequence and track quality metrics, a series of HTGS keywords were introduced. Assembled sequences can be submitted to the CON (contig) division of GenBank using an AGP file .

What is contamination in sequencing?

contamination: All assemblies should be screened for foreign and vector sequences. The source of these foreign sequences can range from bacterial genom e contamination (due to propagating clones in bacteria) to contamination from other projects being sequenced at a particular sequencing center.

What was the first WGS assembler?

The first WGS assemblers, used for bacterial and viral genomes and for BAC clones, were Phrap (Phil Green), TIGR Assembler (Granger Sutton), and Cap3 (X. Huang). These assemblers were widely used during the 1990s.; Some examples of recent WGS assemblers that have been applied successfully to large (mammalian-size) genomes, sequenced using Sanger technology are:

What is the production of a WGS contig?

Figure 2. Production of a WGS contig. These contigs contain no gaps, although the sequence may contain 'N's due to sequence ambiguity. WGS contigs obtain accessions similar to the ones shown in the figure, with the first 4 letters representing a project code, the first two numbers representing the assembly version, and the last 6 numbers providing unique identifiers for each contig.

What is a clone based approach?

The Hierarchical approach (often referred to as 'clone-based') relies on mapping a set of large insert clones (typically BAC or fosmid clones) using methods such as Fingerprint analysis or identifying clones that contain markers localized by linkage mapping or radiation hybrid (RH). Typically, numerous clones will cover any given location of the genome (depending upon the library depth and mapping method used). A minimal tiling path of clones (see figure 1.) is selected in which all sequence is covered with the least amount of redundant sequence produced. Note that there can be substantial overlap between clones. The amount of overlap between clones will vary depending on how the library was constructed.

How many reads are needed to reconstruct DNA?

Each sequenced piece of DNA is referred to as a sequencing read (read for short). Several thousand to several million reads must be produced to reconstruct the sequence of a longer molecule. Both raw reads and assembled data (regardless of the method used) are typically available.

Does polymorphism occur in the genome?

genome polymorphism: Most genomes exhibit some degree of polymorphism. When there is a high level of polymorphism assembly can be confounded. Typically this can lead to either haplotype expansion, in which both alleles are represented in the assembly as separate loci, or representation of a single haplotype in the assembled portion of the genome and of the alternate haplotype in an unplaced contig with no known relationship to the other haplotype.

What is sequence assembly?

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 cannot read whole genomes in one go, but rather reads small pieces of between 20 and 30,000 bases, depending on the technology used.

Why is sequence assembly complex?

The complexity of sequence assembly is driven by two major factors: the number of fragments and their lengths. While more and longer fragments allow better identification of sequence overlaps, they also pose problems as the underlying algorithms show quadratic or even exponential complexity behaviour to both number of fragments and their length. And while shorter sequences are faster to align, they also complicate the layout phase of an assembly as shorter reads are more difficult to use with repeats or near identical repeats.

How many reads can Illumina generate?

From 2006, the Illumina (previously Solexa) technology has been available and can generate about 100 million reads per run on a single sequencing machine. Compare this to the 35 million reads of the human genome project which needed several years to be produced on hundreds of sequencing machines. Illumina was initially limited to a length of only 36 bases, making it less suitable for de novo assembly (such as de novo transcriptome assembly ), but newer iterations of the technology achieve read lengths above 100 bases from both ends of a 3-400bp clone. Announced at the end of 2007, the SHARCGS assembler by Dohm et al. was the first published assembler that was used for an assembly with Solexa reads. It was quickly followed by a number of others.

How many bases are in pyrosequencing?

This new sequencing method generated reads much shorter than those of Sanger sequencing: initially about 100 bases, now 400-500 bases. Its much higher throughput and lower cost (compared to Sanger sequencing) pushed the adoption of this technology by genome centers, which in turn pushed development of sequence assemblers that could efficiently handle the read sets. The sheer amount of data coupled with technology-specific error patterns in the reads delayed development of assemblers; at the beginning in 2004 only the Newbler assembler from 454 was available. Released in mid-2007, the hybrid version of the MIRA assembler by Chevreux et al. was the first freely available assembler that could assemble 454 reads as well as mixtures of 454 reads and Sanger reads. Assembling sequences from different sequencing technologies was subsequently coined hybrid assembly .

Why is Nanopore sequencing important?

Despite the higher error rates of these technologies they are important for assembly because their longer read length helps to address the repeat problem. It is impossible to assemble through a perfect repeat that is longer than the maximum read length; however, as reads become longer the chance of a perfect repeat that large becomes small. This gives longer sequencing reads an advantage in assembling repeats even if they have low accuracy (~85%).

What is EST assembly?

Expressed sequence tag or EST assembly was an early strategy, dating from the mid-1990s to the mid-2000s, to assemble individual genes rather than whole genomes. The problem differs from genome assembly in several ways. The input sequences for EST assembly are fragments of the transcribed mRNA of a cell and represent only a subset of the whole genome. A number of algorithmical problems differ between genome and EST assembly. For instance, genomes often have large amounts of repetitive sequences, concentrated in the intergenic regions. Transcribed genes contain many fewer repeats, making assembly somewhat easier. On the other hand, some genes are expressed (transcribed) in very high numbers (e.g., housekeeping genes ), which means that unlike whole-genome shotgun sequencing, the reads are not uniformly sampled across the genome.

What are short fragments of DNA called?

Typically the short fragments, called reads, result from shotgun sequencing genomic DNA, or gene transcript ( ESTs ). The problem of sequence assembly can be compared to taking many copies of a book, passing each of them through a shredder with a different cutter, and piecing the text of the book back together just by looking at the shredded pieces.

What is a de novo sequence assembler?

De novo sequence assemblers are a type of program that assembles short nucleotide sequences into longer ones without the use of a reference genome. These are most commonly used in bioinformatic studies to assemble genomes or transcriptomes. Two common types of de novo assemblers are greedy algorithm assemblers and De Bruijn graph assemblers.

What is the N50 of a plant genome assembly?

N50 analysis: assemblies by the Plant Genome Assembly Group (using the assembler Meraculous) and ALLPATHS, Broad Institute, USA (using ALLPATHS-LG) performed the best in this category, by an order of magnitude over other groups. These assemblies scored an N50 of >8,000,000 bases.

What are graph method assemblers?

Graph method assemblers come in two varieties: string and De Bruijn. String graph and De Bruijn graph method assemblers were introduced at a DIMACS workshop in 1994 by Waterman and Gene Myers. These methods represented an important step forward in sequence assembly, as they both use algorithms to reach a global optimum instead of a local optimum. While both of these methods made progress towards better assemblies, the De Bruijn graph method has become the most popular in the age of next-generation sequencing. During the assembly of the De Bruijn graph, reads are broken into smaller fragments of a specified size, k. The k-mers are then used as nodes in the graph assembly. Nodes that overlap by some amount (generally, k-1) are then connect by an edge. The assembler will then construct sequences based on the De Bruijn graph. De Bruijn graph assemblers typically perform better on larger read sets than greedy algorithm assemblers (especially when they contain repeat regions).

What is the lowest total genome coverage?

All assemblers performed relatively well in this category, with all but three groups having coverage of 90% and higher, and the lowest total coverage being 78.5% (Dept. of Comp. Sci., University of Chicago, USA via Kiki).

What are the two types of algorithms used by assemblers?

There are two types of algorithms that are commonly utilized by these assemblers: greedy, which a im for local optima, and graph method algorithms, which aim for global optima. Different assemblers are tailored for particular needs, such as the assembly of (small) bacterial genomes, (large) eukaryotic genomes, or transcriptomes.

Genome

Sequencing

Genome Assembly

• Once we have small pieces of the genome, we have to combine (assemble) them together based on their overlap information and build the complete genome. This process is called assembly. Assembly is like solving a jigsaw puzzle. Special software tools called assemblers are used to assemble these reads according to how they overlap, in order to generat...

See more on towardsdatascience.com

Two Main Types of Assemblers

What Can Go Wrong in Genome Assembly?

How to Evaluate Assemblies?

Getting Hands Dirty

How Did They Figure Out The Covid-19 Genome at First?

Final Thoughts

References