Long-Read Sequencing Emerging in Medical Genetics

1Since its introduction over a decade ago, next-generation sequencing (NGS) of DNA has become a routine diagnostic tool for modern medical genetics and revolutionized the discovery of novel Mendelian disease genes. Due to the high-throughput nature and low costs of NGS compared to Sanger sequencing, previous single gene approaches have largely been replaced by gene panels and whole exome sequencing (WES), allowing better clinical diagnostics. However, despite the numerous advancements that NGS has conferred, many studies are still hindered by the short-read lengths (∼150–300 bp) that the current NGS technologies are bound to use in order to preserve high read quality. Issues arising from the use of short reads can mainly be pinned down to the highly repetitive and complex nature of the human genome. It has been shown that despite the use of sophisticated bioinformatic algorithms it is often impossible to accurately map, or even assemble, short reads originating from regions harboring structural variation (SV), repetitive sequences, extreme guanine-cytosine (GC) content, or sequences with multiple homologous elements within the genome. This introduces errors in calling genetic variants and inability to capture certain genomic regions. Furthermore, with short-read NGS (SR-NGS) the variant phasing information is often lost and the data analysis is highly dependent on the reference genomes, which are known to be imperfect. The dependency on the reference genome is especially problematic for the detection of SVs at complex genomic regions that can be highly individual- or population-specific.

2Genetics research has always been strongly driven by novel sequencing technologies, first by Sanger sequencing and later followed by NGS. With the recently demonstrated success in identifying previously intractable DNA sequences and closing gaps in the human genome assemblies, long-read sequencing (LRS) technologies hold the promise to overcome specific limitations of NGS-based investigations of human diseases. The main advantage of LRS compared to other platforms stems from the use of long-reads (>10 kilobase [kb] on average), originating from single DNA molecules. Moreover, the sequencing process occurs in real-time and both the sequencing and library preparation are conducted without the need of PCR amplification, therefore being free from any PCR related bias. The absence of PCR leaves the DNA in its native state, enabling LRS technologies to directly detect base modifications, such as methylation.

3Attributable to these features, LRS has the potential to grow into a technology that is used not only to produce high-quality genome assemblies (i.e., the platinum human reference genome), but also to capture clinically relevant genomic elements which are problematic for conventional approaches (summarized in Figure 1). This has been shown in several studies by (1) identifying disease causing SVs that SR-NGS might not detect, (2) directly sequencing repeat expansions and regions with extreme GC-content, (3) resolving variant phasing and (4) distinguishing a gene of interest from its pseudogenes (Table 1). Importantly, recent studies have indicated that LRS technology may play an important role in discovering novel pathogenic mutations in human diseases with previously unknown genetic causes and open up unprecedented opportunities to investigate transcriptomics. In the future, this technology could ultimately allow whole genome sequencing (WGS) and even de novo assembly capturing all variant types present in individual genomes, independently of the reference genome limitations. In this review, we summarize the LRS-based research of human genetic diseases and discuss the future promise of these technologies to enable the next major advancements in medical genetics. We focus on the prevailing true LRS methods that have been commercially released so far; single molecule real-time (SMRT) sequencing by Pacific Biosciences (PacBio) and nanopore sequencing by Oxford Nanopore Technologies Inc. (ONT) (Box 1 for technical summary).

4True versus synthetic LRS

5The current LRS technologies can be divided into true LRS and synthetic LRS technologies. In synthetic LRS approaches the long stretches of DNA are linked by molecular barcodes, which is followed by sequencing with conventional short-reads and in silico construction of the original DNA molecule. Usually these methods rely on serial-dilutions of very long DNA fragments and physical separation before library preparation in which molecule specific barcodes are added. Similar technologies have recently been further optimized and commercialized by 10× Genomics Inc.. These technologies have already provided haplotype aware SR-WGS and improved SV calling from SR-WGS data. While synthetic long-reads leverage advantages of LRS, some short-read issues may persist, e.g., PCR-bias and intra-read complexity may not always be fully resolved.

6SMRT sequencing by PacBio

7As a first commercial platform for LRS, PacBio released the RS sequencer in 2011, which was followed by RSII in 2013 and Sequel in 2015. SMRT technology is based on special flow cells harboring individual picolitre-sized wells with transparent bottoms. Each of the wells, referred to as zero mode waveguides (ZMW), contain a single fixed polymerase at the bottom. This allows a single DNA molecule, which is circularized in the library preparation (i.e., the SMRTbell), to progress through the well as the polymerase incorporates labeled bases onto the template DNA. Incorporation of bases induces fluorescence that can be recorded in real-time through the transparent bottoms of the ZMW. The average read length for SMRT was initially only ∼1.5 Kb, and with reported high error rate of ∼13% characterized by false insertions. However, the errors are randomly distributed across the reads and high consensus sequences can be obtained with sufficient read depths. Also, for a single molecule, with ∼10 kb read length, each nucleotide position in a 1kb amplicon can be read ∼10 times using circular consensus sequence (CCS) method, rendering it unlikely that a same random mistake would occur in multiple reads. Since its introduction, the read length and throughput of SMRT technology have substantially increased. Throughput can reach >10 Gb per SMRT cell for the Sequel machine, while the average read length for both RSII and Sequel is >10 kb with some reads spanning >100 kb. Still, the persisting downsides for this technology are the high cost/throughput ratio and the requirement of relatively high amounts of high quality DNA as starting material.

8Nanopore sequencing by ONT

9As an alternative, scientist have since many years aimed to use biological or synthetic nanopores to read the sequence of DNA molecules. In 2015, nanopore sequencing was commercially introduced by ONT with a portable MinION sequencer, which was followed by more high-throughput desktop sequencers GridION and PromethION. The basic principle of nanopore sequencing is to pass a single strand of DNA molecule through a nanopore which is inserted into a membrane, with an attached enzyme, serving as a biosensor. Changes in electrical signal across the membrane are measured and amplified in order to determine the bases passing through the pore in real-time. The nanopore-linked enzyme, which can be either a polymerase or helicase, is bound tightly to the polynucleotide controlling its motion through the pore. For nanopore sequencing, there is no clear-cut limitation for read length, except the size of the analyzed DNA fragments. On average, ONT single molecule reads are >10 kb in length but can reach ultra-long for some individual reads lengths of>1 Mb surpassing SMRT. Also, the throughput per run of ONT GridION and PromethION sequencers are higher than for PacBio (up to 100 Gb and 6 Tb per run, respectively). The raw reads have high error rates similar to SMRT dominated by false deletions and in particular homopolymer errors. Currently, the errors are more systematic, and are related to the length and type of the DNA fragment in the nanopore itself, and thus may not all be overcome by just increasing the coverage as with SMRT sequencing.

10The main limitations of current LRS technologies

11Library preparation: for optimal LRS, fresh material or even intact cells are required. The DNA isolation protocols that are needed and the handling of ultra-long high molecular weight DNA require improvements.

12Error rate: both LRS technologies still have a higher error rate compared to SR-NGS. SMRT sequencing may overcome this with CCS also for long-insert libraries. It remains to be seen whether this is sufficient for all variant types including SNVs and indels. So far, ONT has more systematic error-profile, which may be more challenging to overcome.

13Costs: while LRS is still more expensive than SR-NGS, recent advancements in throughput may offer even lower prices. ONT’s PromethION already offers 30× coverage WGS for less than 1,000 dollars, and PacBio’s 8M chip should also significantly reduce the price per human genome. It is also important to realize that the pricings are constantly changing in this rapidly developing field: https://docs.google.com/spreadsheets/d/1GMMfhyLK0-q8XkIo3YxlWaZA5vVMuhU1kg41g4xLkXc/htmlview?hl=en_GB (documentation of updated sequencing costs by Dr. A Vilella).

14Data analysis: both raw data analysis as well as mapping and variant calling tools are much less mature for LRS than SR-NGS but are constantly being improved.

15Whole exome sequencing (WES), mainly using Illumina platforms, is often used as a first-tier test for many genetic diseases. This has been very successful and has brought genetic testing and diagnostics into a whole new era. However, for many patients that have undergone diagnostic WES, or even WGS, the underlying cause of their disease remains unsolved. Curiously, the recent WGS studies applying LRS have revealed that each human genome harbors >20,000 of SVs (>50 bp) and additional thousands of indels (<50 bp), spanning dozens of megabases (Mbs), that have largely remained undetected with conventional SR-NGS. In patients with suspected genetic diseases, such hidden SVs and indels may well disrupt relevant genes or cause a dosage change for dosage-sensitive genes. Furthermore, NGS methods heavily rely on PCR, which leads to GC-content dependent coverage bias. Low or zero coverage at genomic locations with extreme GC-contents are estimated to span >150 Mb of gene-rich regions and some of those may as well account for a proportion of the hidden genetic variation underlying human diseases. Among the approaches that are based on SR-NGS, the PCR-free library preparation methods provide the most uniform genome coverage and may allow sequencing of some of the previously difficult regions. However, SR-NGS always requires a PCR-step in the template amplification, e.g., bridge-PCR, which may still introduce a sequencing bias.

16In contrast to the contemporary SR-NGS methods, LRS technologies require no PCR amplification and sequencing is performed on single molecules instead of colonies or clusters of amplified DNA molecules, resulting in a more evenly distributed coverage. This, together with longer reads that can span over challenging genomic regions and SVs, may offer an unprecedented view on previously poorly characterized genomic locations and eventually improve the variant calling of population-scale sequencing data. While increases in the throughput and lower prices may in the future allow more long-read WGS for individual patients, targeted approaches are already demonstrating the value of LRS for medical genetics (Tables 1, 2).

17High-depth sequencing by NGS is a reliable and cost-efficient method for detecting single nucleotide variants (SNVs) and small indels. However, in the detection of larger genomic deletions, duplications and rearrangements, SR-NGS approaches often lack sensitivity, show excess of false positives and misinterpret complex SVs. Yet, SVs >50 bp in size are an important source of genetic variation, accounting for the greatest number of divergent bases across human genomes. Moreover, SMRT sequencing of a haploid human genome has shown that as much as 89% of the identified variation, consisting mainly of SVs, had been missed in the 1000 genomes project. Similarly, a sevenfold increase in SV detection was achieved by a multi-platform approach, including LRS, compared to standard SR-NGS methods. It is likely that this gain in SV detection sensitivity is due to the different sequencing technology itself, as this benefits from longer fragments, no PCR, and less sequence bias.

18The detection of SVs by SR-NGS is indirect and based on read depth coverages and use of paired-end reads, for which the approximate length between read pairs is known. For copy number variants (CNVs), the excess of reads can indicate amplification whereas the loss of reads is suggestive for a deletion. Rearrangements can be discovered using paired-end reads by investigating deviations in the expected distances and orientations between read pairs. In contrast to SR-NGS, the long reads obtained with PacBio or ONT sequencers are more likely to span SV breakpoints, or often the entire SV event, with high-confidence alignments. Furthermore, longer reads can be more confidently aligned to repetitive sequences that often mediate the formation of SVs. Long reads also allow better distinction of haplotypes, further contributing to the accuracy of analyzing SVs. Other methods, such as microarray copy number profiling and karyotyping, have traditionally been used to detect disease causing SVs. However, with these approaches it is not possible to map small or copy-balanced SVs, and they do not provide accuracy at base-pair resolution, nor the possibility to resolve complex SVs. Thus, LRS can substantially increase the resolution and reliability of SV detection and mapping.

19Recently, proposed LRS as an alternative approach for genome-wide detection of clinically relevant SVs and used ONT-WGS to analyze two patients with congenital abnormalities caused by de novo chromothripsis rearrangements. The authors demonstrated that long reads, even with a relatively low coverage of 16×, are superior to short reads (WGS with average coverage of ∼30×) in detecting and mapping the breakpoints of the rearrangements. The improved phasing capability with long reads also enabled the determination of the parental origin of these de novo events. Other studies have demonstrated the potential benefits of LRS over standard SR-NGS in clinical diagnostics to detect pathogenic SVs. In a study by, a patient with clinically diagnosed Carney complex had previously received negative test results from diagnostic gene panel sequencing and WGS. In contrast, low-coverage SMRT-WGS successfully identified the causative heterozygous deletion of over 2 kb overlapping the first exon of PRKAR1A. Similarly, explored ONT-WGS to confirm the diagnosis for a patient with recessive glycogen storage disease by identifying a second-hit (∼7 kb deletion) in G6PC, which had remained undetected in previous WES analysis. Together, these studies indicate that LRS has the ability to surpass conventional NGS-methods in the detection of SVs and yield clinical diagnoses at a molecular level in previously unsolved cases.

20The fine-mapping of SVs at single-nucleotide resolution is important for patients in which translocation or inversion breakpoints disrupt disease causing genes. For this, long reads are highly beneficial and may render mapping by laborious amplicon-based Sanger sequencing obsolete, as demonstrated by who used SMRT to fine-map BBS9 deletion in a patient with Bardet–Biedl syndrome. In another diagnostic case-study, ONT-WGS was able to track down the exact breakpoints of a reciprocal translocation, which led to the identification of disrupted ARHGEF9 gene in a girl with intellectual disability.

21In addition to the improved identification and mapping of SVs affecting known disease genes, we are now seeing the first discoveries of novel disease genes harboring SVs that are characterized with LRS technologies. As an outstanding example, an unbiased de novo assembly using LRS (SMRT and synthetic longs reads with 10× Genomics) was important for the identification of SINE-VNTR-Alu insertion in TAF1, a newly discovered gene for X-linked Dystonia Parkinsonism. While multiple studies indicate that LRS is superior to SR-NGS in the discovery of SVs, it is also shown that once the alternate allele is resolved, many of these events (∼61%) can be genotyped with high accuracy and lower costs using SR-NGS. This shows that part of the missed SVs are present in the SR-NGS data and therefore improved software may still provide a more complete picture of genomes sequenced with short reads. It is noted that the current cut-off of 50 bp for SVs is arbitrary; indels <50 bp are also identified by LRS and their frequency in WGS is even much higher than for the >50 bp SVs. In addition to PacBio and ONT, synthetic LRS (i.e., by 10× Genomics) can be highly useful in SV detection. Uncovering SVs and indels comprehensively holds the potential to unravel a number of novel disease causing mutations underlying human diseases.

22A short tandem repeat is a region of genomic DNA with multiple adjacent copies of short (1–6 bp) sequence units. These repeat regions are highly mutable due to replication errors that occur during cell divisions and to date over 30 human diseases known to be caused by tandem repeat expansions or repeat contractions. Since their discovery, the research of tandem repeat expansions has been limited by the overall incompliance of these genomic elements to standard molecular techniques like cloning, PCR and sequencing. Most of the disease causing expansions are longer than the currently used NGS reads, making it virtually impossible to accurately assemble those. Multiple studies have now shown that LRS technologies are well suited to transcend through these long, often GC-rich, repeat expansions (Table 1). This not only allows the direct detection of expansion lengths, but also the intra-molecule sequence variation, which might provide clinically relevant additional information.

23The first tandem repeat associated disease studied by LRS was Fragile-X, which is caused by CGG-repeat expansion in the 5′UTR of the FMR1 gene. With standard DNA sequencing technologies, i.e., SR-NGS and Sanger sequencing, it has been impossible to directly sequence the expanded CGG-repeats, consisting of >200 units at full mutation range. SMRT technology enabled for the first time to completely sequence expanded full mutation FMR1 alleles, up to 750 CGG-repeats, which translates to >2 kb of 100% CGG-repeat DNA. The obtained sequencing data and phasing information further allowed to define the presence of ‘AGG’ interruptions, which affect the risk of a premutation to expand into a full mutation in the following generation. The clinical use of this information has been largely neglected due to technical limitations, which can now be overcome with LRS. Genetic variation within repeat expansion loci that are causative for other diseases, such as myotonic dystrophy 1 (DM1) and spinocerebellar ataxia 10 (SCA10), have also been investigated with LRS technology. In DM1, SMRT sequencing detected de novo repeat interruptions at DMPK expansion locus, associated with reduced somatic instability of the repeat expansion and therefore mild or even absent clinical features. Moreover, demonstrated that SMRT sequencing could identify interruption motifs, potentially acting as phenotypic modifiers, within the tandem repeat expansion locus in ATXN10 of SCA10 patients.

24The presented studies on repeat expansions rely on PCR-based target enrichment, which may complicate the analysis due to the occurrence of PCR stutter, chimeric molecules, and false insertions or deletions of the repeat units. Therefore, it has been proposed that the optimal approach to study these repetitive regions would be to sequence single DNA molecules without any prior PCR amplification. For this, have developed CRISPR/Cas9-based amplification-free target enrichment method (No-Amp targeted sequencing, Table 2), which was used to capture repeat the expansion locus in Huntingtin (HTT), responsible for Huntington’s disease. This allowed the retrieval of detailed sequence information and assessment of somatic variability of repeat elements without the interference of PCR stutter. In addition, No-Amp approach has been utilized to successfully sequence across C9orf72 and ATXN10 repeat expansion loci in patients with frontotemporal dementia or Parkinson’s disease, respectively. These studies indicate that No-Amp Targeted sequencing could provide an optimal targeted solution to study different repeat elements that are recalcitrant to PCR-based methods.

25In the era of CRISPR/Cas9 genome editing, LRS has found use in monitoring the efficiency of editing challenging genomic regions. excised a disease causing tandem repeat expansion in the DMPK locus in DM1 patient-derived cell lines and used SMRT sequencing to monitor the efficiency of the excision. In the future, LRS could be more broadly implemented in genome editing of challenging regions. Moreover, with the capability of directly sequencing through long tandem repeats, LRS technology is already accelerating the discovery and characterization of novel repeat expansions linked to human diseases. So far, this has been demonstrated in familial forms of epilepsy, bipolar disorder and schizophrenia. It is noted that, while the current approaches still largely rely on targeted enrichment of suspected repeat expansion loci, ultimately the analysis of all repeat-expansions genome-wide will be retrieved directly by WGS with long reads.

26Phasing refers to the means of assigning genetic variants to the homologous paternal and maternal chromosomes. In medical genetics, phasing is very important for understanding, e.g., inheritance patterns, parental origin of de novo mutations, mosaicism, allele-specific expression and disease risk-haplotypes. However, in order to directly resolve the haplotype of two heterozygous SNVs they would need to be covered by the same molecule (i.e., read). With SR-NGS this is usually only the case for a limited number of variants (20–30% of substitutions), despite the use of paired-end reads. Therefore, phasing has largely been based on parental genotypes and statistical imputation or, in some cases, laborious physical separation of entire chromosomes. Now, with the substantially longer read lengths, LRS technology enables to directly phase variants multiple kbs apart or even assemble and phase complex genomic regions, such as the major histocompatibility complex, in full-length in a diploid human genome.

27Knowing whether two variants in the same gene are in cis or trans can be crucial for diagnostic purposes, especially for compound heterozygosity, which is a commonly observed phenomenon underlying recessive Mendelian disorders. By using LRS, two SNVs in the same gene can be directly phased without the need of testing the parents or if one of the two mutations has occurred de novo and the parental genotypes cannot resolve the phase in the offspring. In pre-implantation genetic diagnostics (PGD), LRS can be applied to determine the parental origin of pathogenic de novo mutations by simply sequencing across the adjacent variants. Resolving the parental origin of de novo mutations is needed for estimating the recurrence risk of a genetic disease in the case of germline mosaicism. LRS could also be used in PGD to directly phase parental genotypes for genome-wide haplotype reconstruction of a single cell via discrete SNP genotypes (i.e., haplarithmisis). Moreover, phasing with long reads offers an accurate method to study mosaicism. This has recently been shown by who used SMRT to resolve allele phasing in an exceptional case of keratitis-ichthyosis-deafness syndrome exhibiting revertant mosaicism, a rare phenomenon involving spontaneous correction of a pathogenic mutation by another mutation in a somatic cell. To keep in mind, pointed out problems in resolving allele phasing with targeted SMRT amplicon sequencing. In their study, the formation of chimeric molecules during PCR-amplification led to unambiguous results, which could have been avoided by using PCR-free methods. However, these methods can be costly and time consuming and simply keeping the amount of PCR cycles as low as possible could offer an easier solution.

28Identification of SVs may also benefit from phasing, in particular SV calling in cancer genomes can be significantly improved to understand the complexity of chromosomal rearrangements. Furthermore, phasing with LRS can be used to reveal the subclonal heterogeneity in malignancies. For genome-wide association studies (GWAS), multi-allelic haplotype markers can provide superior power compared to single-SNP markers in mapping disease loci. However, the implementation of haplotype-based GWAS is highly dependent on capability to phase alleles. Therefore, LRS could be utilized to fine-map genetic associations within regions of interest that have previously been identified by GWAS. In addition to these multiple situations where accurate phasing is of great importance, LRS technology and the emerging ultra-long reads (>100 kb) from latest LRS developments may eventually produce fully phased diploid genomes, which can even originate from single cells. This would allow for research that fully leverages phase information in order to understand how phenotypes are influenced by unique haplotype combinations.

29Pseudogenes usually originate from gene duplication or retrotransposition events and are defined as sequences that resemble known functional genes but cannot produce functional proteins. Notably, the GENCODE project has estimated that the human genome contains more than 14,000 pseudogenes and many clinically relevant genes (such as PMS2, CYP2D6, CHEK2, SMN1, and PKD1) are known to have pseudogenes with high sequence homology. This has significant consequences for re-sequencing studies as pseudogenes may severely impair reliable variant identification in their functional counterparts and lead to false diagnostic test results. Traditional methods, such as SR-NGS may capture sequences from pseudogenes in addition to the functional genes, leading to mapping errors, low variant detection rates and high number of false positives. Other issues originate from the fact that some pseudogene sequences are still missing from the human reference genome and may cause mapping issues for SR-NGS. Therefore, assays based on SR-NGS may not always be reliable for investigating genes that have highly homologous pseudogenes.

30To date, several studies (Table 1) have utilized LRS in order to investigate clinically relevant genes with homologous pseudogenes. These studies have largely relied on target enrichment with long-range PCR (LR-PCR) using primers that locate to the rare mismatch sites that distinguish the gene of interest from its pseudogenes. Similar approaches have been used with Sanger sequencing and SR-NGS. However, with LRS, the reads can span the complete LR-PCR amplicon and retain the phase information. Furthermore, reference-free assembly of long reads can circumvent errors that may be introduced by complex or repetitive regions in the reference genome. For example, Polycystin 1 (PKD1), responsible for autosomal dominant polycystic kidney disease, has traditionally been challenging to analyze with NGS or Sanger sequencing because of its high GC-content, large size and homology with pseudogenes. demonstrated that by coupling LR-PCR with SMRT, the interference of residual PKD1 pseudogene amplification and alignment ambiguities could be eliminated, and SVs could be identified. In addition, multiple studies have demonstrated the utility of LRS for genotyping and phasing CYP2D6, which is one of the most important genes involved in drug metabolism, but has been difficult to analyze by conventional methods due to homologous pseudogenes and complex gene rearrangements.

31For medical genetics research, it is crucial to have reliable information on the population frequencies of particular gene variants in order to assess their potential pathogenicity. For this, databases such as ExAC and GnomAD are often used. However, reference datasets attained with SR-NGS might be problematic for genes with highly homologous pseudogenes. This is mainly because: (1) the deposited variants may be derived from pseudogenes, (2) true variants might stay undetected or (3) the gene of interest might be ambiguously covered. Long reads could be used to improve the annotation of duplicated regions because they harbor sufficient number of paralogous sequence variants to confidently assign them to their respective paralogs. Therefore, in addition to more reliable pseudogene discrimination, LRS can be used to generate more accurate reference datasets and annotations for many challenging genes, subsequently allowing improvements in variant interpretation.

32The emergence of NGS, together with the incessancy of technological advances, drives our increasing ability to uncover complex and previously ‘hidden’ genetic aberrations, however, our current capacity to interpret their functional and clinical impact remains restrained. By providing a global transcriptomic profile, both quantitatively, i.e., gene expression levels, and qualitatively, i.e., transcript sequences and isoform structures, RNA sequencing holds the potential to reduce this diagnostic gap significantly. Recently two research groups pioneered this technology as a complementary tool to DNA-based tests and demonstrated its value in the detection of both coding and non-coding variants. In particular, authors identified a variety of variants underpinning (1) abnormal or mono-allelic expression and (2) aberrant splicing, resulting in the creation, skipping and truncation of exons, as well as exon and intron retention, even in regions harbouring repetitive regions. In turn, resulting in a diagnostic yield ranging from 10% for mitochondriopathies to 21% for primary muscle disorders in patients lacking strong candidates from WES or WGS.

33Similar to NGS-based genomic approaches, however, short-read RNA sequencing methodologies are limited by their need for computational reconstruction of individual short reads into complete transcripts. With the advent of long-read RNA sequencing the accurate identification and quantification of full-length mRNA transcript isoforms has become possible (Figure 2). A comparative approach by has revealed up to 17% of novel alternative exons to be detected following long-read RNA sequencing of three primary human tissues, i.e., brain, heart, liver, thereby corroborating the isoform landscape to be more complex than initially thought. In addition, the authors demonstrate the applicability of full-length mRNA sequencing to uncover transcriptional regulation and post-translational modification profiles, including alternative transcription initiation, allele specific alternative splicing and alternative 3′ termination (polyadenylation). The clinical significance of integrating this transcriptomic data was exemplified by, who identified an intronic retrotransposon to induce alternative splicing that subsequently affects TAF1 expression, in turn causing X-linked Dystonia-Parkinsonism. Moreover, by exploring long-read RNA sequencing in the characterization of premature termination codons in ABCA7 in late-onset Alzheimer’s disease, observed various degrees of nonsense-mediated decay (NMD) and transcript modification that potentially influence ABCA7 dosage and disease severity. While the ultimate goal may be to employ this technique to allocate the causative variant and map its functional impact on a patient-level, there may already be a major improvement by sequencing reference sets of tissue/cell specific RNA-isoform landscapes. Gaining insight into the isoform landscape of disease relevant tissues or cell types, potentially even on a single-cell level, will advance the overall interpretation of clinical significance of complex rearrangements, tissue-specific isoforms and splice variants. Ultimately, this knowledge can be implemented to improve WES/WGS-based variant filtering, prioritization and prediction of their functional impact.

34Most of the clinically relevant examples described in this review use targeted LRS approaches, indicating that the broader use of LRS could significantly increase the diagnostic yield of genetic testing and discover novel disease genes. Especially SVs, repetitive elements and complex genomic regions that are difficult to assess with short reads can now be better assessed. LRS technology is also changing our view on the mRNA isoform landscape of different tissues and genes, potentially enabling better functional interpretation of genomic variation in the future. It is noted that, while the currently used LRS technologies are not quite yet at the stage of individual fully phased de novo assembled genomes, but with increased throughput, higher accuracy and lower costs, LRS-based WGS is coming within reach. Once this is routine, we are foreseeing the possibility that WGS with long reads could serve as a truly generic test that enables to detect all genetic variants present in an individual’s genome. Then the power of LRS to overcome the current limitations in medical genetics may be elucidated at fast pace. Moreover, systematic studies utilizing LRS in patient cohorts with unsolved genetic diseases and control populations are warranted, and several consortia, e.g., www.solve-rd.eu or http://www.internationalgenome.org/1000-genomes-browsers, have already announced the use of LRS-WGS for this.

35While the medical genetics community is now starting to realize the potential of this technology, some obstacles need to be overcome in order for LRS to be established as a mainstream tool (for overview of LRS limitations, see Box 1). Especially library preparation and analysis still need to reach the high level of robustness of SR-NGS technologies. For the data analysis, bioinformatic tools have traditionally been optimized for short-read sequencing data and need to be adapted, as well as thoroughly tested, for long reads. In addition, analyzing whole genomes with LRS is still very expensive and the future application will depend largely on pricing progress of all NGS technologies, and may also depend on how fast the short-reads can reach an even lower prices with even better quality (i.e., the ∼100$ genome). Eventually, one might need to consider a choice between sequencing many less-expensive genomes with short-reads as opposed to investigating a smaller number of genomes in-detail using long reads. It is not a straightforward decision to make, however, the latter option would ideally be beneficial for an individual patient in clinical diagnostics. Combining SR-NGS and LRS can also be a powerful approach for highly accurate variant calling and assembly and become increasingly important and more easily available with the planned acquisition of PacBio by Illumina.

36We foresee that both of the prevailing LRS technologies, PacBio and ONT, will have a major influence on the future of medical genetics. ONT has the potential to become low in price, ease of use technology that is capable of directly investigating both native DNA and RNA in a high-throughput manner. The main challenge for ONT technology is to circumvent the systematic error rate, e.g., for homopolymer regions, which may be a limitation especially for future diagnostic applications. ONT has put forward ideas to improve this by optimizing bioinformatic tools that allow more accurate repeat lengths measurements and proposed to leverage different types of nanopores. One possible improvement was already enabled by the rolling circle amplification to concatemeric consensus method (R2C2). For SMRT sequencing, the consensus accuracy achieved is better, especially once intra-molecule consensus – in addition to the existing inter-molecule consensus – calling is also feasible for long-insert libraries. To enable human genome sequencing, throughput needs to go up. To this end, PacBio has recently announced to release a SMRT cell with 8 million ZMWs in 2019 (∼8-fold increase) and other improvements may come from even longer read lengths. This would enable much higher throughput and reduction in sequencing costs. Altogether, it remains to be seen whether the quality of the LRS technologies is sufficient for genome-wide detection of small indels and SNVs. If so, PacBio and ONT will have a chance to become the platform for true WGS for population scale studies in the near future. Moreover, the native epigenetic modifications of the DNA are preserved in PCR-free single molecule sequencing, opening up possibilities for novel LRS-based applications to assess base modifications.

37In addition to already commercially available technologies discussed in this review, others are in active development and will hopefully be available for users in the future. These include novel nanopore-based technologies from Genia (now acquired by Roche) and Stratos Genomics, or the Electronic Nano-Device sequencing (ENDSeq) from Roswell Biotechnologies. Other exciting technologies that may also provide long-read insights are, e.g., library-and amplification-free technology from Nanostring (Hyb & Seq), nanochannel genome mapping technology from Bionano Genomics and Strand-Seq that preserves long-range context of homologous chromosomes. Whether these or other technologies are complementary to the existing LRS approaches, or may be able to compete with or even replace PacBio and ONT remains to be seen.

38To conclude, we are on the advent of next revolution in sequencing technology. Once the major obstacles regarding accuracy, analysis and pricing are overcome, the time would be right to move towards individual fully phased long-read genomes, likely based on individual de novo assemblies that enable the identification of all the genetic variants regardless of their type.

39AH and TM conceived the study. TM, SK, and AH drafted manuscript. SK prepared the figures.

40The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.