The third generation sequencing: The advanced approach to genetic diseases

The third generation sequencing: The advanced approach to genetic diseases

Abstract: Genomic sequencing technologies have revolutionized mutation detection of the genetic diseases in the past few years. In recent years, the third generation sequencing (TGS) has been gaining insight into more genetic diseases owing to the single molecular and real time sequencing technology. This paper reviews the genomic sequencing revolutionary history first and then focuses on the genetic diseases discovered through the TGS and the clinical effects of the TGS, which is followed by the discussion of the improvement in the bioinformatic analysis for the TGS and its limitations. In summary, the TGS has been enhancing the diagnostic accuracy of genetic diseases in molecular level as well as paving a new way for basic researches and therapies.

Keywords: Diagnostic; genomics; third generation sequencing (TGS); genetic disease

第三代测序:基因疾病的先进方法
文摘:
在过去的几年中，基因组测序技术已经彻底改变了基因疾病的突变检测。
近年来，由于单分子实时测序技术的应用，第三代测序技术(TGS)对更多的遗传疾病有了更深入的了解。
本文首先回顾了基因组测序的革命历史，然后重点介绍了通过TGS发现的遗传疾病和TGS的临床效果，然后讨论了TGS在生物信息学分析方面的改进及其局限性。
综上所述，TGS在分子水平上提高了遗传疾病诊断的准确性，为基础研究和治疗开辟了新的途径。
关键词:诊断;基因组学;第三代测序(TGS);遗传性疾病

IntroductionOther Section

The definition of a rare disease is a disease affecting fewer than 2,000 people in Europe (1), while, a disease affecting less than 200,000 people is defined as a rare disease in the United States. Though the chance that individuals who are diagnosed with each rare disease is seemingly low, approximately 7,000–8,000 rare diseases are estimated to date. The Global Genes Project estimates that 300 million people worldwide are affected by a rare disease and eighty percent of rare diseases are gene of origin (2). Moreover, about fifty percent of those affected by rare diseases are in their childhood, thirty percent of who will not survive beyond their fifth year old.

The current genomic technological advancement has changed the research approaches and clinical strategies of rare diseases. In the past few years, the human genome project was firstly completed in mapping all the genes in human with a cost of almost 3 billion dollars. Then the sequencing price significantly dropped with the application of the next generation sequencing (NGS). High throughput and low cost of genomic sequencing make the further insight into genetic diseases in more patients possible. However, the percentage of all known rare diseases with the pathogenic gene is less than fifty percent (3). One reason is that the routine sequencing technology has missed some mutations. Hence, it has still been a challenge to develop diagnostics, managements and genetic advice for these patients in practice.

This paper aims to provide a review of the third generation sequencing (TGS) in genetic diseases. A brief review of revolution of the sequencing technology in genetic diseases is firstly presented. And then, an overview of the TGS approach to the genetic diseases and its clinical effect follows. The bioinformatic methods applied to the new technology and limitations of the TGS are also discussed.

介绍其他部分
在欧洲，罕见病的定义是指感染人数少于2000人的疾病(1)，而在美国，感染人数少于20万人的疾病被定义为罕见病。
尽管每个人被诊断出患有每种罕见疾病的几率似乎很低，但迄今估计大约有7000到8000种罕见疾病。
全球基因工程估计全世界有3亿人受到一种罕见的疾病,百分之八十的罕见疾病基因的起源(2)。此外,约百分之五十的罕见疾病的影响在他们的童年,百分之三十的人将无法生存超过他们五岁。
当前基因组技术的进步改变了罕见病的研究方法和临床策略。
在过去的几年里，首次完成了人类基因组计划，绘制了人类所有的基因图谱，耗资近30亿美元。
随着下一代测序技术的应用，测序价格明显下降。
基因组测序的高通量和低成本使进一步了解更多患者的遗传疾病成为可能。
然而，所有已知的罕见病中含有致病基因的比例不到50%(3)。原因之一是常规测序技术漏掉了一些突变。
因此，在实践中为这些患者开发诊断、管理和遗传建议仍然是一个挑战。
本文就基因疾病的第三代测序(TGS)进行综述。
首先简要回顾了基因疾病测序技术的革命。
然后，对遗传疾病的TGS方法及其临床效果进行综述。
讨论了生物信息学方法在新技术中的应用以及TGS的局限性。

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Brief revolution of sequencing technology Other Section

Aside from the study of genetic diseases via karyotyping, DNA microarrays, FISH or multiples ligation-dependent probe amplification (MLPA), the first generation sequencing, including Maxam Gilbert methods and Sanger sequencing opened up the new door into the genetic diseases since 1977.

The next generation sequencing (NGS) emerged in 2004 helped researchers gain deeper understanding about the genetic diseases (4). More than 2,400 pathogenic genes have been identified (5) and over 150 genetic diseases have been identified via the whole exome sequencing (WES) (6). Three important centers for Mendelian Genomics (CMGs) funded by NIH, including University of Washington, Yale University and the Baylor College of Medicine utilized the NGS to elucidate many Mendelian disorders (7). In short, the NGS has a great impact on de novo mutations in rare diseases in recent years. However, many rare diseases are still not fully diagnosed by the NGS due to the short-read methods (~150–300 bp). Structural variants (SVs), repetitive elements, extreme guanine-cytosine (GC) content or sequences with multiple homologous elements in the genome are difficult to be characterized via the NGS, even with the use of state-of-the-art bioinformatic algorithms (8,9). These drawbacks of the NGS-based investigations of human diseases have strongly driven the search for other methods to improve the accuracy and reduce diagnosing time in genetic diseases.

The TGS provided by Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) in 2011, is a single molecular and real-time sequencing technology (10).The PacBio platform adopts single-molecule real-time (SMRT) technology (Figure 1). In the DNA library preparation, no PCR is required as a closed and circular ssDNA template can be replicated automatically. During the sequencing process, the fluorescence signals are activated by a laser as soon as a labeled dNTPs is incorporated into DNA. A camera system then records the color and duration of the emitted light in real time in the flow cell equipped with zero mode waveguides (ZMVs). The time of the base incorporation is longer as the base is modificated. Thus, the time called “interpulse duration” can indicate the DNA modification event (Figure 2) (11). The SMRT technology also allows the direct RNA-sequencing (12). The SMRT essentially is still based on sequencing by synthesis. Nanopore Sequencing Technology (ONT) utilizes nanopore inserted in an electrical resistant membrane. A potential is applied across the membrane, resulting in a current flowing only through the nanopore (Figure 3). The characteristic disruptions in the current can be measured, indicating a specific single molecular. In the DNA library preparation, a hairpin structure is designed to ligate the double DNA strands so that the system can read both DNA strands in one continuous read. As dsDNA moves through the nanopore, the bound polymerase or helicase enzyme can attach the DNA in the pore. During sequencing process, a characteristic disruption in the electrical current can be measured as the nucleotide passing through the nanopore (Figure 4) (13). Then the nucleotide can be identified. These features allow the detection of hundreds of kilobases in one continuous read. Ultra-long reads (ULRs) with above 300 kb reads and some close to 1 million bp reads can be sequenced in the ONT (14). Also, the many pocket-sized sequencers developed by the ONT are portable without sophisticated laboratory setup and can be transported out of the lab with low cost. For example, the MinION was transported to Africa for screening Ebola and Lassa virus outbreak (15,16). In short, the features of the TGS introduced by PacBio and ONT allow for the long-read sequencing in real-time with the low alignment and mapping errors during library construction.

简要革命的测序技术其他部分
除了通过核型分型、DNA微阵列、FISH或多次连接依赖探针扩增(MLPA)研究遗传疾病外，自1977年以来，包括Maxam Gilbert方法和Sanger测序在内的第一代测序为遗传疾病的研究打开了新的大门。
下一代测序(上天)出现在2004年帮助研究人员获得更深的理解(4)的遗传疾病。2400多个致病基因已经被鉴定(5)和150多基因疾病已确定通过全外显子组测序(韦斯)(6)。三个重要孟德尔基因组学中心(发生)由国家卫生研究院资助,包括华盛顿大学,
耶鲁大学(Yale University)和贝勒医学院(Baylor College of Medicine)利用NGS阐明了许多孟德尔疾病(Mendelian disorders)(7)。总之，NGS对近年来罕见疾病的新生突变有很大的影响。
然而，由于NGS的短读方法(~ 150300 bp)，仍有许多罕见病未被NGS充分诊断。
即使使用最先进的生物信息算法，结构变异(SVs)、重复元素、极端鸟嘌呤胞嘧啶(GC)含量或基因组中有多个同源元素的序列也很难通过NGS来表征(8,9)。
基于ng基的人类疾病研究的这些缺陷强烈推动了对其他方法的研究，以提高遗传疾病的准确性和缩短诊断时间。
2011年由太平洋生物科学公司(PacBio)和牛津纳米孔技术公司(ONT)提供的TGS是一种单分子实时测序技术(10)。
PacBio平台采用单分子实时(SMRT)技术(图1)。在DNA文库制备中，不需要PCR，可以自动复制一个封闭的、圆形的ssDNA模板。
在测序过程中，一旦标记的dNTPs进入DNA，就会用激光激活荧光信号。
然后，一个摄像系统在配备了零模波导(ZMVs)的流池中实时记录发出光的颜色和持续时间。
随着碱基的修改，碱基掺入的时间变长。
因此，称为脉冲间持续时间的时间可以表示DNA修饰事件(图2)(11)。
SMRT技术还允许直接rna测序(12)。
SMRT本质上仍然是基于合成的排序。
纳米孔测序技术(ONT)利用纳米孔插入电阻膜。
在膜上施加一个电势，导致电流只流经纳米孔(图3)。电流的特征中断可以被测量，表明一个特定的单个分子。
在DNA文库制备中，设计了发夹结构将双链DNA连接起来，使系统能够一次连续读取两条DNA链。
当dsDNA穿过纳米孔时，结合的聚合酶或解旋酶可以附着在小孔中的DNA上。
在测序过程中，当核苷酸通过纳米孔时，可以测量出电流的特征中断(图4)(13)。
然后核苷酸就可以被识别了。
这些特性允许在一次连续读取中检测数百个kb。
超过300 kb读取和接近100万个bp读取的超长读取(ulr)可以在ONT中测序(14)。
此外，ONT开发的许多口袋大小的测序仪无需复杂的实验室设置就可携带，并且可以以低成本运输出实验室。
例如，仆从被运送到非洲筛查埃博拉和拉沙病毒爆发(15,16)。
简而言之，PacBio和ONT引入的TGS的特性使得在构建库的过程中，可以实时进行长读测序，低比对和映射误差。

Figure 1 SMRT sequencing (image adapted from PACIBO website). SMRT, single-molecule real-time.

Figure 2 A methylated base sequenced by the PacBio; Interpulse duration (dotted arrow in Figure 2A) [image adapted from reference (11)]. PacBio, Pacific Biosciences.

Figure 3 Nanopore sequencing and current signals (Image adapted from Oxford Nanopore Technologies website).

Figure 4 A methylated base (red) sequenced by the ONT. [Image adapted from reference (13)]. ONT, Oxford Nanopore Technologies.

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Comprehensive genetic disease identification Other Section

The genetic disease can be understood in molecular level rather than that of chromosome owing to the development of sequencing technologies. The TGS has not only helped to discover more novel genetic diseases (17), but also revised the identification of genetic disease (17).

SV has an important role in genetic disorders (18). SVs are defined as mutations affecting more than 50 base pairs. The SVs include deletions, insertions, inversions, mobile element transpositions, translocations, tandem repeats and copy number variants (CNVs) (19). By using SMRT sequencing for two haploid human genomes, Huddleston’s group pointed out that estimated approximately 89% SVs have been missed in the 1,000 Genomes Project (20). Although the sophisticated SV genotyping software methods were available, the detection of SVs was low (30–70%) and the error rates were still high (85%) (21). The single molecular and real-time sequencing has shown a better capacity to discover the structural-variant events. A few SVs related genetic diseases detected through the TGS is reviewed in the following part. For example, Aneichyk and colleagues studied X-linked Dystonia-Parkinsonism (XDP) which is a Mendelian neurodegenerative disease and suggested that a SINE-WNTR-Alu (SVA) mediated aberrant transcriptional mechanism was associated with XDP (22). The precise breakpoints of the deletion in a homozygous 7p14.3 were deciphered in the proband with Barde-Biedl syndrome (BBS) and carrier parents by long-read SMRT sequencing (23). The WES yielded only one heterozygous causal variants in the patient with glycogen storage disease type Ia (GSD-Ia) which is an autosomal recessive disease (24), while, a 7.1 kb deletion covering two exons in G6PC on the other allele were detected through Nanopore long-read whole genome sequencing (WGS) (24). Multiple neoplasia and cardiac myxoma with the negative NGS results were found in connection with a heterozygous 2,184 bp deletion of the first coding exon of PRKAR1A (25). A complex novel translocation t(X;20)(q11.1;p13) was delineated via Nanopore long read sequencing (LRS) technology in a balanced reciprocal translocation (BRT) case (26). Other congenital diseases associated with complex chromothripsis were identified to link to the de novo complex SV breakpoints via ONT (27). In addition, fine-mapping of dipeptidyl-peptidase 6 gene (DPP6) in an autosomal dominant dementia family significantly linked to 7q36 was identified via the PromethION sequencing platform (Oxford Nanopore Technologies) (28).

Another advantage of the TGS is characterize the characterization of complete repeat expansion of genes and discriminate pseudogenes. As a typical example, the C9ofr72'GGGGCC' (G4C2) repeat expansion associated with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) was validated through Pacific Biosciences and Oxford Nanopore Technologies (29). The CGG short tandem repeats in fragile X syndrome were detected by SMRT sequencing (30). Likely, the familial myoclonic epilepsy was connected with a 4.6 kb repeat expansion and 12.4 kb deletion in complex repeat regions via SMRT (31,32). Additionally, repeat expansions of complex genes, such as ATXN10, HTT, SMAD12, TNRC6A and RAPGEF2 were also validated by SMRT sequencing (33-36). CTG-repeat expansion was confirmed by SMRT in CRISPR/Cas9-mediated editing in myotonic dystrophy patient as well (37). Other complex and challenging regions of the human genome were characterized via the TGS, such as autosomal-dominant polycystic kidney disease (ADPKD). Duplicated and high GC content genomic regions as well as six pseudogenes of PKD1 gene can lead to ambiguous identification of variants via the NGS. However, 94.7% of the patients with PKD1 pathogenic variants were identified via SMAT by Borras and colleagues (38). A GC-rich 60 basepair variable number of tandem repeat (VNTR) and all variants position of the Mucin-1 gene in autosomal dominant tubulointerstitial kidney disease (ADTKD) were also determined by SMRT sequencing (39). Another example is the primary immunodeficiency-associated gene IKBKG. The pseudogene IKBKGP1 can be bypassed by long read, single-molecule sequencing which allows the rapid and efficient identification of the primary immunodeficiency diseases (40). Sanna Gudmundsson and colleagues clarified the mechanism of revertant mosaicism through SMRT (41). They demonstrated that the dominant negative effects of the p.Asp50Asn mutation was reverted by the second-site mutations of Cx25-Asp50Asn resulting in the development of healthy-looking skin in a patient with ichthyosis-deafness (KID) syndrome.

As stated above, the features of the TGS also allows the detection of the epigenetic modification in real time. DNA modifications have been found in a wide range of living organisms, from prokaryotes to eukaryotes. Many existing studies have shown that they play important roles in development diseases, such as lysosomal storage disorders, tumorigenesis, autoinflammatory diseases, imprinting and X chromosome inactivation (42-45). The bisulfite Sanger sequencing and other next generation sequencing have the restriction to read length of only 150–130 bp (46,47). Therefore, long-read single-molecule real-time bisulfite sequencing (SMRT-BS) developed by Yang and colleagues is a technique that combines bisulfite conversion with the TGS and allowed the detection of the targeted CpG methylation in real time (48).

Furthermore, LRS allows the detection of full-length mRNA transcript in one read. The short-read RNA sequencing always leads to inaccurate annotation due to computational transcript reconstruction (49). Aneichyk and colleagues utilized the long-read RNA-sequencing to decipher the TAF1 expression in the X-linked dystonia-parkinsonism (XDP) (22). Roeck and colleagues demonstrated that the Alzheimer’s disease severity was in relation to the varying degrees of nonsense-mediated mRNA decay (NMD) and transcript-modifying events (50). Twenty-seven genetically unsolved patients with an external collagen VI-like dystrophy were found in connection with highly recurrent de novo intronic mutation in COL6A1 via RNA-sequencing (51).

The combination of the TGS with other technologies, such as the NGS or single cell sequencing or target genome editing, will also give an insight into the genome and bring the new therapies (52). Mimori and colleagues utilized the SMRT sequencing and additional short-read data to obtain the high-quality and full-length human leukocyte antigen alleles reconstruction successfully (53). A study of Hendel A and colleagues showed that SMRT sequencing was facilitated to quantify the genome editing outcomes after the large genes were inserted at the endogenous IL2RG, HBB, and CCR5 loci by transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs) or clustered regularly interspaced short palindromic repeats (CRISPR/Cas9 or RNA-guided endonucleases (RGENs) (54).

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Clinical effect of the TGS Other Section

Importantly, clinical decisions and outcomes can be benefited from the TGS applications with more complete detection of mutations. For example, de novo mutations can occur in the different stage of embryonic development. Depending on the different stage of postzygotic mutation during development, such mutations may lead to somatic or germline mosaicism or both (55). Understanding of the complex SVs guided the genetic counseling and enable a successful preimplantation genetic diagnosis in the family (56). Maria and colleagues demonstrated that less than 1% of the TCOF1 variant c.3156C>T cells were in the paternal germ cells in a family with a child suffering Treacher Collins syndrome, suggesting the low recurrence risk in offspring (55). Similarly, there were 40% of PTPN11 variant c.923A>C cells in the paternal germ cells in a family with unsuccessful pregnancies, indicating a high recurrence risk of Noonan syndrome in offspring (55). Moreover, AGG interruptions in females with a FMR1 premutation were detected by long-read single-molecule sequencing, which was previously undetected due to the technical difficulties (57). In short, apart from the increasing discovery of novel disease genes, the TGS aids the preimplantation genetic counseling.

Ethics is also important in gene sequencing technology. The informed consent, data privacy and return of results are three issues demanding attention (58). To date, the recommendations of ethical considerations have been addressed by the American College of Medical Genetics and Genomics (ACMG) (59). Obviously, more ethical issues await the TGS as more discoveries of the novel disease genes in clinical practice come up.

其他科室的临床效果
重要的是，临床决策和结果可以受益于TGS应用更完整的突变检测。
例如，新生突变可能发生在胚胎发育的不同阶段。
根据发育过程中合子后突变的不同阶段，这些突变可能导致体细胞嵌合体或种系嵌合体或两者都嵌合体(55)。
对复杂SVs的理解指导了遗传咨询，使家族成功地进行了植入前遗传诊断(56)。
Maria和同事证明，在患有Treacher Collins综合征的孩子的家庭中，只有不到1%的TCOF1变异c.3156C>T细胞存在于父亲的生殖细胞中，这表明后代的复发风险很低(55)。
同样，在妊娠不成功的家庭中，有40%的PTPN11变异C . 923a>C细胞存在于父代生殖细胞中，表明后代努南综合征的高复发风险(55)。
此外，通过长读单分子测序可以检测到FMR1预突变女性的AGG中断，此前由于技术困难未被检测到(57)。
简而言之，除了不断发现新的疾病基因外，TGS还有助于植入前的遗传咨询。

伦理在基因测序技术中也很重要。
知情同意、数据隐私和结果返回是需要关注的三个问题(58)。
到目前为止，美国医学遗传学和基因组学学院(ACMG)已经提出了伦理考虑的建议(59)。
显然，随着临床实践中对新疾病基因的更多发现，更多的伦理问题等待着TGS。

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Bioinformatic methods in the TGS Other Section

With more discoveries of novel SVs, repeat expansions and long noncoding RNAs (IncRNAs) via the TGS, the bioinformatic algorithms have to be TGS-specific and more user-friendly. The major bioinformatic challenges of the TGS is the high sequencing error rate which is 10–15% in the PacBio and 5–20% in the ONT. Therefore, the new alignment and error correction algorithms are required (Table 1). Several studies have offered the relatively new methods to correct the sequencing errors in the TGS. The methods for alignment and phasing are LAST (77), BLASR (73), BWA-MEM (74), GraphMap (75), MECAT (64) and minimap2 (78), PBHoney (79), NGMLR (74), Sniffles (74), CORGi (83), SIVM (84), SMRT-SV (95), NextSV (96), NanoSV (97) and Picky (98) in de novo mutations and SVs detection. Regarding the RNA sequencing analysis, the available bioinformatic tools include SQANTI (85), TAPIS (86), ToFU (87), BLAT (88), Gmap (89). In terms of errors correction in sequencing analysis, there are a few available methods as well. Hybrid error correction methods including Nanocorr, MaSCA, PBcR or Spades utilize short-read data to correct the error. However, because of biases in short-read coverage and repetitive sequence, FALCON-sense, HGAP, pbCR, Canu or MARVEL is more accurate as they are self-error correction methods (99). The other technique developed by Jana Ebler and colleagues combined the inference of haplotype and genotypes from noisy long reads (100). A similar software named as NanoSim is a fast and large-scale read simulator to call reads errors in MinION platforms (101). A TGS tool developed by Danze Chen’s group is a bioinformatic suit to compare isoforms, identify alternative splicing pattern and IncRNA (102). Moreover, a time and resource effective strategy for completing short read assembles has been applied, which enable sufficient analysis date to be assembled with the shortest sequencing time (103).

生物信息学方法在TGS的其他部分
随着新的SVs、重复扩增和通过TGS发现的长非编码rna (IncRNAs)的增多，生物信息算法必须是针对TGS的，并且更加用户友好。
TGS的主要生物信息学挑战是高测序错误率，在PacBio中错误率为10-15%，在ONT中错误率为5-20%。
因此，需要新的比对和纠错算法(表1)。一些研究提供了相对新的方法来纠正TGS中的测序误差。
在新突变和SVs检测中，采用LAST(77)、BLASR(73)、BWA-MEM(74)、GraphMap(75)、MECAT(64)和minimap2(78)、PBHoney(79)、NGMLR(74)、Sniffles(74)、CORGi(83)、SIVM(84)、SMRT-SV(95)、NextSV(96)、NanoSV(97)和selective(98)等方法进行比对和分期。
在RNA测序分析方面，现有的生物信息学工具有SQANTI(85)、TAPIS(86)、ToFU(87)、BLAT(88)、Gmap(89)。
在测序分析的误差校正方面，也有一些可用的方法。
混合误差校正方法包括Nanocorr, MaSCA, PBcR或Spades利用短读数据来校正误差。
然而，由于短读报道和重复序列的偏差，FALCON-sense、HGAP、pbCR、Canu或MARVEL更准确，因为它们是自错校正方法(99)。
另一项由Jana Ebler及其同事开发的技术结合了单倍型和基因型的推断，这些推断来自于嘈杂的长读数(100)。
一个类似的名为NanoSim的软件是一个快速和大规模的读取模拟器，用于调用MinION平台中的读取错误(101)。
由陈丹泽的团队开发的TGS工具是一种生物信息学套装，用于比较异型，识别替代剪接模式和IncRNA(102)。
此外，还应用了一种时间和资源有效的策略来完成短读组装，这使得以最短的测序时间(103)组装足够的分析数据。

Table 1 Bioinformatic methods in the TGS
Full table

The TGS comes with several limitations (14,43,44). First, the DNA library required fresh material or intact cells and the protocols for the handing of ultra-long high molecular weight DNA require improvements. Second, the TGS has the challenges with the higher sequencing error rate and systematic error. Third, the cost of the TGS still has been higher than that of the NGS ($65–$200 per Gb in the PacBio and $22–$90 per Gb in the ONT). Additionally, because the database systems for interpreting complicated SVs are rare, thus the bioinformatic analysis are challenging.

Currently, the NGS is still our first choice of diagnosing the genetic diseases in clinical settings and the TGS can play a complementary role as a result of its limitations. However, with the maturation of the TGS approach, it will be widely used in researches and clinical practice. In the future, the picture of human genome will be more comprehensive as the more genomic data generated.

TGS有几个限制(14,43,44)。
首先，DNA文库需要新鲜的材料或完整的细胞，处理超长高分子量DNA的协议需要改进。
其次，TGS具有较高的测序错误率和系统错误率。
第三，TGS的成本仍然高于NGS(在PacBio中为65 - 200美元/ Gb，在ONT中为22 - 90美元/ Gb)。
此外，由于用于解释复杂SVs的数据库系统很少，因此生物信息分析具有挑战性。

目前，NGS仍是我们临床诊断遗传病的首选，而TGS由于其局限性，可以起到补充作用。
但随着TGS方法的成熟，将会在研究和临床实践中得到广泛应用。
在未来，随着产生更多的基因组数据，人类基因组的图景将会更加全面。

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AcknowledgmentsOther Section

Thanks for Xinran Dong for the critical reading of the “Bioinformatic methods in the TGS” part of the manuscript.

Funding: None.

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FootnoteOther Section

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/tp.2020.03.06). The authors have no conflicts of interest to declare.

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