What Are the Benefits and Disadvantages of Long Reads Scaffolding Techniques?
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Abstract
In recent years long-read technologies have moved from being a niche and specialist field to a bespeak of relative maturity likely to feature often in the genomic landscape. Analogous to next generation sequencing, the toll of sequencing using long-read technologies has materially dropped whilst the instrument throughput continues to increase. Together these changes present the prospect of sequencing large numbers of individuals with the aim of fully characterizing genomes at high resolution. In this article, we will endeavour to nowadays an introduction to long-read technologies showing: what long reads are; how they are distinct from short reads; why long reads are useful and how they are being used. We volition highlight the contempo developments in this field, and the applications and potential of these technologies in medical research, and clinical diagnostics and therapeutics.
When Brusk Reads Are Not Enough
Deoxyribonucleic acid is an extraordinarily compact storage medium, so minor that developing ways to decode the sequence encoded in these molecules has been a topic of research for many years. The first method developed for sequencing DNA, often known every bit Sanger sequencing (i), was a low throughput procedure that detected bases by incorporation into a template strand, sequencing fragments of Dna up to k bp long. The quantum assuasive sequencing at scale finally came with the advent of next generation sequencing (NGS) applied science, which employed massively parallel reactions for high throughput. While these technologies accept been able to capture sequence from the majority of the genome and take found utility in the study of illness, their brusk reads and lack of contextual information has limited their utility in genome assembly and in resolving complex and repetitive regions of the genome.
The incremental improvements in read-length that this generation of engineering science can yield is one of diminishing returns. Thus, to achieve substantial gains in mapping, assembly and phasing one must consider applied science that provides an order of magnitude increase in read-length (2). Practically likewise, there are many important bug in genetics where a curt read of Dna (<thou base of operations pairs) is insufficient (Table 1 and Fig. 1).
Table i.
Advantages and applications of long-read sequencing
| Limitations of short read data | Applications and advantages of long-read sequencing |
|---|---|
|
|
| Limitations of brusque read information | Applications and advantages of long-read sequencing |
|---|---|
|
|
a MHC: Major histocompatibility complex.
b HLA: Histocompatibility leucocyte antigen.
c ADPKD: Autosomal-dominant polycystic kidney affliction.
Tabular array 1.
Advantages and applications of long-read sequencing
| Limitations of short read information | Applications and advantages of long-read sequencing |
|---|---|
|
|
| Limitations of brusque read data | Applications and advantages of long-read sequencing |
|---|---|
|
|
a MHC: Major histocompatibility complex.
b HLA: Histocompatibility leucocyte antigen.
c ADPKD: Autosomal-ascendant polycystic kidney disease.
Figure i.
Behaviour of reads around genomic events. (A) Large insertion: short reads at the edge of the variant are be soft-clipped. Reads within the insertion will exist either unmapped or mapped incorrectly. Large reads will either span the insertion or have plenty context to be marked equally inserted sequence. (B) Large deletion: short reads spanning the deletion may exist mismapped or only have ane of the reads marked as mapped considering the reference measured length indicates the insert size deviates from the expected distribution. Long reads will span the gap but most will accept plenty context to call the deletion. (C) Copy number variation: where the read-length exceeds the length of the CNV region reads will map correctly. Shorter reads may be collapsed and show up every bit increased depth in a pileup or be marked as mapping poorly. (D) Inversion: reads will either be represented as a primary alignment with an inverted supplementary or manifest as soft clipping around the border of the inversion with a reduction in depth where reads span the edge of the inversion.
Effigy ane.
Behaviour of reads effectually genomic events. (A) Large insertion: curt reads at the edge of the variant are be soft-clipped. Reads within the insertion will exist either unmapped or mapped incorrectly. Large reads will either bridge the insertion or have plenty context to be marked equally inserted sequence. (B) Big deletion: brusque reads spanning the deletion may be mismapped or but have one of the reads marked as mapped considering the reference measured length indicates the insert size deviates from the expected distribution. Long reads volition bridge the gap but most will accept enough context to telephone call the deletion. (C) Copy number variation: where the read-length exceeds the length of the CNV region reads will map correctly. Shorter reads may be collapsed and prove up every bit increased depth in a pileup or be marked every bit mapping poorly. (D) Inversion: reads volition either exist represented as a primary alignment with an inverted supplementary or manifest as soft clipping around the edge of the inversion with a reduction in depth where reads span the edge of the inversion.
Key to achieving high quality results with all long-read technologies is the employ of loftier molecular weight DNA equally a starting cloth. The utility of these methods depends on a long DNA fragment size, with Deoxyribonucleic acid damage and fragmentation limiting the quality of data obtained. Specific protocols for Dna extraction such equally the agarose gel protocol for BioNano are platonic to maximize yield from these methods.
Long-Read Technologies
Single molecule existent fourth dimension sequencing
The first long-read sequencing technology to achieve a widespread deployment is the single molecule existent time (SMRT) sequencing technology from Pacific Biosciences (PacBio). The SMRT system implemented in their Sequel and RS- Two platforms uses a massively parallel arrangement of polymerases each bound to a single molecule of target Dna that has been circularized with a pair of hairpin sequencing adaptors (the SMRTbell) (Fig. 2A). Incorporation of labelled bases past a polymerase on the template strand causes fluorescence. The resulting indicate is detected by a CCD camera via a cipher-fashion waveguide (vi,7), yielding a combination of indicate and time series information. Reads produced by this applied science typically peak at 100 Kbp in length and a typical N50 on recent polymerases is ∼xx Kbp.
Figure 2.
Long-read sequencing technologies. (A) PacBio SMRT sequencing. Double stranded DNA is first sheared and size selected to the desired length and and so sequencing adaptors are annealed. The adaptors are bound to a sequencing primer and strand displacing polymerase which adheres to the bottom of a well containing a nil way moving ridge guide. Following a pre-extension menses where the polymerase reaction is run in the dark, the fragment is illuminated with a laser and as each base in the sequencing solution is incorporated, the fluorophore is detected and the polymerase reaction displaces it, giving a time and intensity signal which is converted into a base of operations call. (B) Oxford Nanopore Engineering passes the DNA molecule through a nanopore fastened the flow cell surface membrane. Every bit each base of the Deoxyribonucleic acid molecule passes through the pore changes to the current passing through the pore are detected and converted into a signal. The betoken detected is passed to a recurrent neural network (RNN) which converts information technology into base calls. (C) 10X Genomics Chromium technology works past means of an emulsion droplet technology, where gel beads are mixed with high molecular weight genomic Deoxyribonucleic acid and an enzyme. Within each gel bead Deoxyribonucleic acid is sheared and barcoded, creating fragments which tin can and then be sequenced with Illumina sequencing. The presence of the chromium barcode then provides a mapper or assembler with linked-reads, assuasive the relative spatial position of the fragments to be estimated Components of figure reproduced with permission from Pacific Biosciences, Oxford Nanopore Technologies and 10X Genomics.
Figure ii.
Long-read sequencing technologies. (A) PacBio SMRT sequencing. Double stranded DNA is kickoff sheared and size selected to the desired length and and then sequencing adaptors are annealed. The adaptors are spring to a sequencing primer and strand displacing polymerase which adheres to the bottom of a well containing a goose egg mode wave guide. Post-obit a pre-extension period where the polymerase reaction is run in the night, the fragment is illuminated with a laser and as each base of operations in the sequencing solution is incorporated, the fluorophore is detected and the polymerase reaction displaces it, giving a time and intensity signal which is converted into a base call. (B) Oxford Nanopore Technology passes the Deoxyribonucleic acid molecule through a nanopore attached the flow prison cell surface membrane. As each base of the DNA molecule passes through the pore changes to the current passing through the pore are detected and converted into a point. The indicate detected is passed to a recurrent neural network (RNN) which converts it into base calls. (C) 10X Genomics Chromium technology works by ways of an emulsion droplet applied science, where gel beads are mixed with high molecular weight genomic Dna and an enzyme. Within each gel bead Deoxyribonucleic acid is sheared and barcoded, creating fragments which tin can then be sequenced with Illumina sequencing. The presence of the chromium barcode and then provides a mapper or assembler with linked-reads, allowing the relative spatial position of the fragments to be estimated Components of figure reproduced with permission from Pacific Biosciences, Oxford Nanopore Technologies and 10X Genomics.
One complication of SMRT sequencing is the high error rate of this process relative to short read sequencing, at xi–fourteen% depending on polymerase and chemistry. Notwithstanding, this fault style is stochastic (by contrast with other technologies), and can be mitigated by repeated measurements of the sequence. With PacBio sequencing, this is carried out by repeated forward and reverse sequencing passes over the circularized SMRTbell molecule (Fig. 2A). Adaptor sequences tin can be removed from the generated sequence to provide plenty subreads to generate a highly accurate consensus of each molecule. This process is known as round consensus sequencing and has been shown to reduce basecalling error substantially (eight) whilst likewise enabling the strand specific calling of base of operations modifications in unamplified DNA (9). When long DNA fragments are sequenced, these may not be parsed more than once in the SMRTbell; in this case, increasing coverage and so calling a consensus across reads can also attain a reduced error rate; a method ofttimes used in polishing assemblies (x).
Oxford Nanopore Technologies
The next successful single molecule applied science to striking the marketplace was that produced past Oxford Nanopore Technologies (ONT) (xi). This engineering science is based on passing a single strand of DNA through a nanopore with an enzyme attached, and measuring changes in the electrical signal across the pore (Fig. 2B). The signal is then amplified and measured to determine the bases that passed through. As the pore holds several bases at a time (typically 5-mers), overlapping k-mers that cause changes in raw current must be inferred and used to make base of operations calls, a procedure which can be error decumbent. Past measuring the shape of the molecule passing through the pore ONT not only reads the sequence of the Dna only like SMRT is also able to find base modifications (12). However, unmodelled base modifications and systematic Deoxyribonucleic acid context-specific errors (13) currently limit the utility of the technology.
Oxford Nanopore MinION technology heralds the promise of a pocket size sequencer, with reads from ONT that can stretch into the hundreds of kilobases with appropriate DNA preparation, and megabase long reads that have been observed when a large number of menstruation cells have been used. In that location appears to be no intrinsic read-length limit for ONT, other than the size of Dna fragments. Recent improvements in engineering science, library grooming and throughput have allowed the first human line sequenced on the MinION (GM12878) earlier this twelvemonth (14). This written report generated ultra-long reads (>800 Kbp), and suggested that addition of small coverage with ultra-long-read sequencing to existing assemblies may substantially improve resolution of contigs and haplotypes. While the mistake charge per unit is comparable to SMRT sequencing, a component of the fault is systematic and context-specific, limiting the ability to right this by increasing coverage (13) and requiring polishing with other technologies instead.
ONT has adult a distinct strategy to mitigate stochastic mistake on their platform, focusing on the way that the template strand passes through the pore. ONT cannot simply circularize the DNA. Instead, both the template and complement strands of the Dna molecule are joined by a hairpin loop during library prep (2d) or tethered in such a fashion (1Dii) to permit sequential forwards and opposite strand sequencing. Combining these information greatly enhances accuracy and reduces random fault.
The utilize of nanopores as a nucleic acid sequencing technology is not entirely sectional to ONT; at least one similar but distinct competing technology is too under development by Roche.
10X Genomics Chromium organisation
An alternative to the aforementioned single molecule sequencing methods is the 10X Genomics Chromium system. Whilst this is non technically a long-read sequencing technology, it is an important member of this ecosystem and tin can solve similar issues such equally mapping, phasing and assembly (Fig. 2C). Chromium has lower cost compared to ONT and SMRT because of the employ of the nearly ubiquitous Illumina short reads in its sequencing procedure.
The basis of this technology (15) is the barcoding of large fragments of DNA (preferably >100 Kbp) in an initial digital droplet polymerase chain reaction (PCR) step. In each droplet, a single fragment is both sheared and then tagged with a semi-unique molecular barcode (Fig. 2C). The resulting fragments are and then sequenced similar any other Illumina library. The barcode allows for determination of the relative spatial orientation of the tags, and allows phasing and assembly of contigs by combining information across multiple tags (xv,16). Additionally, because the data provide spatial orientation across the genome, it is possible to use it to scaffold data from other methods (17).
Centrolineal technologies
Centrolineal technologies associated with long-read sequencing such as: optical mapping, HiC and like accept been used to enhance the final results from sequencing. Optical mapping technologies such as BioNano Irys and Saphyr characterization DNA and then image the labelled Deoxyribonucleic acid to generate genome maps. These genome maps are used to scaffold contigs produced past assembly (18) and as well to discover large (>500 bp) structural variants and inversions. HiC tin be used to analysis chromosomal conformation and is particularly useful in assigning assembled sequences to chromosomes (19).
The Utility of Long-Read Technology: Recent Developments
High resolution genome assemblies
Accurate assemblies of the genomes of organisms are crucial to agreement organismal diversity, speciation, evolution of species and the impact of genomic multifariousness on health and disease. The electric current human genome reference GRCh38 has been assembled from the DNA of multiple donors, and represents a mosaic of haplotypes. However, several studies take suggested that existing human reference genomes may not fully reflect the diversity of global homo populations, and may be biased towards diversity in European populations (twenty–22). This has of import implications for human basic and medical enquiry. Assembling the human genome has involved extensive curation with clone-based assembly methods and Sanger sequencing. Long-read technologies provide a high throughput platform for characterization of genomes through highly contiguous assemblies (Fig. 3).
Figure 3.
Long reads span and call variations that short reads cannot. IGV (http://software.broadinstitute.org/software/igv/home) image of (top) PacBio reads from a sample sequenced as part of the GDAP projection. The reads bridge a 6 kb heterozygous LINE-1 element deletion and show clear depth variation. Illumina (bottom) reads from the aforementioned sample unable to be clearly mapped effectually the deletion with reads in white indicating where reads were unable to be uniquely mapped.
Figure three.
Long reads span and telephone call variations that curt reads cannot. IGV (http://software.broadinstitute.org/software/igv/home) prototype of (top) PacBio reads from a sample sequenced as part of the GDAP project. The reads bridge a 6 kb heterozygous LINE-1 element deletion and show articulate depth variation. Illumina (bottom) reads from the same sample unable to exist clearly mapped around the deletion with reads in white indicating where reads were unable to exist uniquely mapped.
The early long-read platforms produced reads that were only a few kilobases long with a high per-base price; however, they chop-chop carved a niche in the creation and finishing of assemblies. These long reads could close gaps in genomes by spanning the low complexity regions that would otherwise require many plush YAC, BAC and fosmid clones to be created and sequenced. Thus, many of the early on tools such as PBJelly were focused on gap closure (23,24). The high per-base error rate besides required new assembly algorithms, and new tools were created to polish the final associates with Illumina reads to eliminate basecalling error (12). Clone based assembly methods were non eliminated entirely either every bit they provided useful spatial context, but long reads provided a new mode to sequence clones in a high throughput manner (25).
Long-read sequencing methods have contributed to platinum quality reference sequences such as NA12878 (xiv,26) and the haploid sequences CHM1 (27) and CHM13 (28), as well filling many gaps in the human reference (18,25,29). Of item notation are the first Chinese (18) and Korean (25) human reference genomes which have been created to reply questions about population-specific sequence. These sequences have resulted in highly contiguous assemblies, endmost a high proportion of gaps in the human genome. These take led to discovery of population-specific sequences, demonstrating the need for further assemblies from non-European population groups. Recently, higher coverage sequencing (∼60×) of two haploid genomes has also been used to place substantial structural variation, the vast bulk of which take not been recovered from sequencing using NGS technologies (28). Characterization of high resolution population-specific reference genomes from initiatives such every bit the Genome in a Bottle (GIAB) (30) and the Genome Variety in Africa project (GDAP) (31) (Fig. three) will provide of import resources for population and medical genetics, and too allow a clearer understanding of the evolutionary demographic history of different populations by better delineation of phase (31).
Most human assemblies accept involved a haploid representation of the genome, where information from the ii chromosomes is collapsed into a single sequence. Generation of haplotype representations of the genome can reduce error in the final assembly, particularly in the case of segmental duplications (xvi,32). While long-read technologies can generate phase information over long contiguous segments, these methods cannot resolve phase over long regions of homozygosity or assembly gaps. Assembly of haploid genomes, therefore, requires boosted contextual information, which can be provided by linked-read approaches. More recently, trio based methods (where parents are sequenced using Illumina short reads, with offspring sequenced with long reads) have been used to provide this contextual information by separation of maternal and paternal haplotypes prior to assembly using a father–mother–offspring trio (33). This method has been practical to yield a highly contiguous diploid assembly of an F1 hybrid of two bovine subspecies with a quality surpassing previous cattle reference genomes (33).
Long reads have been successfully applied to organisms with smaller genomes besides every bit bacteria and viruses, with the advantage that for some of these the entire genome can be spanned by a single long read (34). The Tree of Life initiative, a collaboration across multiple centres is in the process of developing loftier resolution reference sequences for >50 vertebrate species using a combination of long read, brusk read and linked-read approaches. Some other leading project is the big bacterial sequencing projection NCTC 3000 at the Wellcome Sanger Institute, which is using PacBio sequencing to sequence consummate bacterial genomes (https://www.phe-culturecollections.org.uk/collections/nctc-3000-project.aspx). These relatively small genomes (Escherichia coli is for example 4.6 Mbases) tin can often have their chromosomes and plasmids assembled into unmarried contigs. The construction of full and authentic assemblies of these organisms allow fine-scale phylogenies of these organisms to be constructed and is too helpful in the field of epidemiology when tracing the source of an outbreak. A contempo example of this was a study where SMRT sequencing was used to identify a reservoir of antibiotic resistant plasmids inside hospitals (35).
In improver to Dna sequencing, ONT sequencing has been applied to sequence RNA directly rather than relying on an intermediate cDNA stride, allowing direct sequencing of RNA viruses and detection of splice variants and base modifications straight from RNA molecules. An instance of this is the contempo directly sequencing and assembly of the influenza A virus in a native RNA form without amplification or conversion to DNA (36).
Targeted sequencing
From a clinical point of view targeted sequencing is an area where long reads are likely to have the greatest initial bear on. In the diverse, complex and clinically relevant regions such as the histocompatibility leucocyte antigen (HLA) (37), killer prison cell immunoglobulin-like receptor (KIR) (38) and BRCA; and in pharmacologically relevant genes such every bit CYP2D6 (39,xl), targeted sequencing has allowed clinicians and researchers to characterize areas of the genome which were previously inaccessible using NGS methods. In addition, where diversity is high it has become possible to call and phase variation across the entire gene. This approach has since been used to retype 126 HLA reference samples across 6 loci and is now considered a gilt standard for clinical sequencing for stem cell transplants (41).
Typically, when targeting such a region, a long-range PCR reaction is used to specifically dilate the genes of interest. Yet recently there accept been studies demonstrating the use of pulldowns and CRISPR-CAS9 to capture the region of interest with little or no distension (42). The reward of these reduced and non-amplification based approaches is the removal of PCR fault as a factor, specially in tandem repeats and GC rich regions (42). Additionally, in the instance of CRISPR methods, capture of raw genomic textile allows DNA modification information to be read.
Transcriptomics and RNA
In add-on to its many uses with Deoxyribonucleic acid, long-read technology also has provided many new insights into the world of transcriptomes and ncRNA past assuasive for sequencing of these total length isoforms rather than relying on the associates of sheared NGS fragments, a method prone to a high charge per unit of false positives and ambiguities (43). Direct sequencing of isoforms tin can be particularly useful in circuitous polyploid genomes such every bit the coffee institute (44), where construction of a reference transcriptome is otherwise extremely challenging. In add-on to its usefulness in reference transcriptomes IsoSeq has been used in functional studies to analyse the expression of diverse disease-linked proteins such as TP53 in leukaemia (45).
The MinION platform has recently been used to sequence cDNA; applications of this, such as single cell sequencing of allowed cells illustrates the power of such methods to examine clonal heterogeneity in gene expression and isoform usage, potentially revolutionizing our agreement of the repertoire and functions of immunological cell receptors (46).
Epigenetics
SMRT sequencing technology is able to detect base modification, as it records base kinetics of the polymerase, when Deoxyribonucleic acid molecules are sequenced directly without PCR. Similarly, Nanopore technology tin also detect base modifications due to variation in ionic currents. However, because distension of DNA would erase base modifications, these methods require relatively large amounts of native, unamplified Deoxyribonucleic acid as input material. Contempo innovations that combine bi-sulphate conversion with SMRT sequencing have immune direct loftier throughput analysis of CpG methylation without requiring large quantities of sample (47), providing an artery for more accurate assessment of CpG islands, and allele-specific CpG methylation.
Clinical applications
The advantages of long-read technologies in accessing complex regions of the genome, make these ideal for clinical applications in diagnosis, prognostication and personalized medicine. Early clinical applications have included sequencing of tandem repeats in fragile X syndrome, spinocerebellar ataxia, providing authentic diagnostics and potential for prognostication in clinical genetics. SMRT sequencing has besides been used to resolve structural variants associated with Mendelian disease (48).
Long-read sequencing technologies are quickly moving towards the mainstay of high resolution HLA typing for transplant registries in certain regions (37); with high resolution typing potentially having implications for meliorate matching, and clinical outcomes of patients undergoing transplantation. This is even more important in populations which are poorly represented in electric current reference sequence databases, limiting disambiguation of clinical types when using standard methods for typing. The HLA diversity in Africa projection, which aims to characterize high resolution HLA types across >20 ethno-linguistic in Africa has recently completed sequencing of ∼2000 individuals using long-read sequencing, identifying high levels of novelty in class I and grade Ii HLA types (49). This panel volition provide an important resources for clinical HLA typing in populations of African ancestry, as well as a platform for highly accurate imputation of HLA types in medical genetics research.
Using long-range PCR amplicons, with barcoding and long-read technology besides allow better delineation of genes from pseudogenes, such as for sequencing PKD1 for diagnosing autosomal-dominant polycystic kidney disease, for which diagnostic accuracy of NGS technologies has been express (50). SMRT sequencing has also been used to tailor treatment in patients with cancer, by identifying low frequency resistant mutations in BCR-ABL1 that bear on treatment efficacy in patients with CML (51). Applications of SMRT sequencing in reproductive medicine, to place parent of origin effects, and for pre-implantation diagnosis accept been previously noted (52).
Full sequencing of several virus genomes in a unmarried contig by long-read sequencing has provided unique avenues for identification of resistant mutations for clinical applications. Proof-of concept studies take generated protocols to examine low frequency (up to 0.25%) associated mutations for HIV and HCV resistance to drugs, through deep sequencing of full length quasispecies (53). Methylation profiles of pathogens examined using SMRT approaches have also been shown to correlate with pathogenicity, and virulence, potentially providing a new avenue for applications in communicable diseases surveillance.
The Future
Long-read technologies are improving speedily, and may get the mainstay of sequencing; however, the broader awarding of long-read technologies are currently limited by a lower throughput, college error rate and higher toll per base relative to short read sequencing. Wider employ of such technologies in the clinical context may apace improve our understanding of cancer, pathogen evolution, drug resistance and genetic diversity in circuitous regions of the genome that take important implications for clinical intendance. Parallel development of existing technology to allow high throughput PCR-free sequencing volition be important in sequencing difficult regions of the genome (54).
At nowadays, no single long-read technology has any articulate advantage from a scientific point of view, and thus it seems probable that the time to come of long-read sequencing is more likely to exist decided on commercial terms rather than scientific. Whichever technology captures the marketplace, it is clear that as these technologies become more than affordable they will proceed to shine a calorie-free into previously intractable regions of the genome with ever larger sample sizes and longer read-lengths, assuasive new discovery in these evolving fields.
Acknowledgements
Uttara Partap for copyediting.
Conflict of Involvement statement. M.O.P has presented at a PacBio-sponsored meeting and has received accommodation for presenting at this effect.
Funding
Wellcome Trust (grant number 098051 to M.S.South.); the National Plant for Wellness Research Cambridge Biomedical Research Centre (Great britain) to Chiliad.Due south.S.; a Wellcome Trust Fellowship (grant number 106289/Z/xiv/Z to A.J.Thou.); and the Medical Research Council (MRC) (MR/S003711/1 to D.G.); IAVI with the generous support of USAID (in part), and the Bill & Melinda Gates Foundation; a full list of IAVI donors is bachelor at www.iavi.org. The contents of this manuscript are the responsibility of the authors and practice not necessarily reflect the views of USAID or the U.s. Government. Funding to pay the Open Access publication charges for this article was provided past the Wellcome Trust.
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