Recurrent neural network for predicting absence of heterozygosity from low pass WGS with ultra-low depth.

BACKGROUND: The absence of heterozygosity (AOH) is a kind of genomic change characterized by a long contiguous region of homozygous alleles in a chromosome, which may cause human genetic disorders. However, no method of low-pass whole genome sequencing (LP-WGS) has been reported for the detection of AOH in a low-pass setting of less than onefold. We developed a method, termed CNVseq-AOH, for predicting the absence of heterozygosity using LP-WGS with ultra-low sequencing data, which overcomes the sparse nature of typical LP-WGS data by combing population-based haplotype information, adjustable sliding windows, and recurrent neural network (RNN). We tested the feasibility of CNVseq-AOH for the detection of AOH in 409 cases (11 AOH regions for model training and 863 AOH regions for validation) from the 1000 Genomes Project (1KGP). AOH detection using CNVseq-AOH was also performed on 6 clinical cases with previously ascertained AOHs by whole exome sequencing (WES). RESULTS: Using SNP-based microarray results as reference (AOHs detected by CNVseq-AOH with at least a 50% overlap with the AOHs detected by chromosomal microarray analysis), 409 samples (863 AOH regions) in the 1KGP were used for concordant analysis. For 784 AOHs on autosomes and 79 AOHs on the X chromosome, CNVseq-AOH can predict AOHs with a concordant rate of 96.23% and 59.49% respectively based on the analysis of 0.1-fold LP-WGS data, which is far lower than the current standard in the field. Using 0.1-fold LP-WGS data, CNVseq-AOH revealed 5 additional AOHs (larger than 10 Mb in size) in the 409 samples. We further analyzed AOHs larger than 10 Mb, which is recommended for reporting the possibility of UPD. For the 291 AOH regions larger than 10 Mb, CNVseq-AOH can predict AOHs with a concordant rate of 99.66% with only 0.1-fold LP-WGS data. In the 6 clinical cases, CNVseq-AOH revealed all 15 known AOH regions. CONCLUSIONS: Here we reported a method for analyzing LP-WGS data to accurately identify regions of AOH, which possesses great potential to improve genetic testing of AOH.

Identifying potential drug targets for idiopathic pulmonary fibrosis: a mendelian randomization study based on the druggable genes.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic fibrotic interstitial lung disease characterized by progressive dyspnea and decreased lung function, yet its exact etiology remains unclear. It is of great significance to discover new drug targets for IPF. METHODS: We obtained the cis-expression quantitative trait locus (cis-eQTL) of druggable genes from eQTLGen Consortium as exposure and the genome wide association study (GWAS) of IPF from the International IPF Genetics Consortium as outcomes to simulate the effects of drugs on IPF by employing mendelian randomization analysis. Then colocalization analysis was performed to calculate the probability of both cis-eQTL of druggable genes and IPF sharing a causal variant. For further validation, we conducted protein quantitative trait locus (pQTL) analysis to reaffirm our findings. RESULTS: The expression of 45 druggable genes was significantly associated with IPF susceptibility at FDR < 0.05. The expression of 23 and 15 druggable genes was significantly associated with decreased forced vital capacity (FVC) and diffusing capacity of the lungs for carbon monoxide (DLco) in IPF patients, respectively. IPF susceptibility and two significant genes (IL-7 and ABCB2) were likely to share a causal variant. The results of the pQTL analysis demonstrated that high levels of IL-7 in plasma are associated with a reduced risk of IPF (OR = 0.67, 95%CI: 0.47-0.97). CONCLUSION: IL-7 stands out as the most promising potential drug target to mitigate the risk of IPF. Our study not only sheds light on potential drug targets but also provides a direction for future drug development in IPF.

Validation and depth evaluation of low-pass genome sequencing in prenatal diagnosis using 387 amniotic fluid samples.

BACKGROUND: Low-pass genome sequencing (LP GS) is an alternative to chromosomal microarray analysis (CMA). However, validations of LP GS as a prenatal diagnostic test for amniotic fluid are rare. Moreover, sequencing depth of LP GS in prenatal diagnosis has not been evaluated. OBJECTIVE: The diagnostic performance of LP GS was compared with CMA using 375 amniotic fluid samples. Then, sequencing depth was evaluated by downsampling. RESULTS: CMA and LP GS had the same diagnostic yield (8.3%, 31/375). LP GS showed all copy number variations (CNVs) detected by CMA and six additional variant of uncertain significance CNVs (>100 kb) in samples with negative CMA results; CNV size influenced LP GS detection sensitivity. CNV detection was greatly influenced by sequencing depth when the CNV size was small or the CNV was located in the azoospermia factor c (AZFc) region of the Y chromosome. Large CNVs were less affected by sequencing depth and more stably detected. There were 155 CNVs detected by LP GS with at least a 50% reciprocal overlap with CNVs detected by CMA. With 25 M uniquely aligned high-quality reads (UAHRs), the detection sensitivity for the 155 CNVs was 99.14%. LP GS using samples with 25 M UAHRs showed the same performance as LP GS using total UAHRs. Considering the detection sensitivity, cost and interpretation workload, 25 M UAHRs are optimal for detecting most aneuploidies and microdeletions/microduplications. CONCLUSION: LP GS is a promising, robust alternative to CMA in clinical settings. A total of 25 M UAHRs are sufficient for detecting aneuploidies and most microdeletions/microduplications.

Comprehensive interpretation of single-nucleotide substitutions in GJB2 reveals the genetic and phenotypic landscape of GJB2-related hearing loss.

Genetic variants in GJB2 are the most frequent cause of congenital and childhood hearing loss worldwide. The purpose of this study was to delineate the genetic and phenotypic landscape of GJB2 SNV variants. All possible single-nucleotide substitution variants of the coding region of GJB2 (N = 2043) were manually curated following the ACMG/AMP hearing loss guidelines. As a result, 60 (2.9%), 177 (8.7%), 1499 (73.4%), 301 (14.7%) and 6 (0.3%) of the variants were classified as pathogenic, likely pathogenic, variant of uncertain significance, likely benign, and benign, respectively. 53% (84/158) of the pathogenic/likely pathogenic missense variants were not present in ClinVar. The second transmembrane domain and the 310 helix were highly enriched for pathogenic missense variants, while the intracellular loops were tolerant to variation. The N-terminal tail and the extracellular loop showed high clustering of variants that are associated with syndromic or dominant non-syndromic hearing loss. In conclusion, our study interpreted all possible single-nucleotide substitution coding variants, characterized novel clinically significant variants in GJB2, and revealed significant genotype-phenotype correlations at this common hearing loss locus. Our work provides a prototype for other genes with similarly high genetic and phenotypic heterogeneity.

The Next Generation of Population-Based DFNB16 Carrier Screening and Diagnosis: STRC Copy-Number Variant Analysis from Genome Sequencing Data.

BACKGROUND: Deafness, autosomal recessive 16 (DFNB16) is caused by compound heterozygous or homozygous variants in STRC and is the second most common form of genetic hearing loss. Due to the nearly identical sequences of STRC and the pseudogene STRCP1, analysis of this region is challenging in clinical testing. METHODS: We developed a method that accurately identifies the copy number of STRC and STRCP1 using standard short-read genome sequencing. Then, we used whole genome sequencing (WGS) data to investigate the population distribution of STRC copy number in 6813 neonates and the correlation between STRC and STRCP1 copy number. RESULTS: The comparison of WGS results with multiplex ligation-dependent probe amplification demonstrated high sensitivity (100%; 95% CI, 97.5%-100%) and specificity (98.8%; 95% CI, 97.7%-99.5%) in detecting heterozygous deletion of STRC from short-read genome sequencing data. The population analysis revealed that 5.22% of the general population has STRC copy number changes, almost half of which (2.33%; 95% CI, 1.99%-2.72%) were clinically significant, including heterozygous and homozygous STRC deletions. There was a strong inverse correlation between STRC and STRCP1 copy number. CONCLUSIONS: We developed a novel and reliable method to determine STRC copy number based on standard short-read based WGS data. Incorporating this method into analytic pipelines would improve the clinical utility of WGS in the screening and diagnosis of hearing loss. Finally, we provide population-based evidence of pseudogene-mediated gene conversions between STRC and STRCP1.

Using 24-h intraocular pressure-related patterns to identify open-angle glaucoma in thyroid eye disease.

PURPOSE: Our study aims to develop a diagnostic model using 24-h intraocular pressure (IOP) patterns to differentiate between open-angle glaucoma (OAG) and dysthyroid optic neuropathy (DON) in thyroid eye disease (TED) patients with glaucoma-like symptoms. METHODS: TED patients with elevated IOP, abnormal optic disc, and/or visual fields were prospectively recruited. The subjects whose symptoms were relieved by DON first-line treatments were divided into the DON group, and the subjects with previous diagnosis of OAG before TED onset were divided into the OAG group. The 24-h IOP was monitored by Tono-Pen in a sitting position during awake time and in a supine position during sleep time. All subjects were divided into a training set and a testing set. The diagnostic models were generated from training set by using either IOP curve-derived parameters or principal component (PC) factors. The discrimination ability was tested in training set based on area under curve (AUC), and the calibration ability was verified in testing set by Hosmer-Lemeshow goodness-of-fit. The sensitivity and specificity were calculated by two-by-two table with the cutoff value determined by Youden's index. RESULTS: Thirty-two cases were recruited in each group. The 24-h IOP curves revealed a nocturnal pattern in both groups, with the acrophase moving slightly forward in the DON group (21:00 pm-24:00 pm) compared to the OAG group (22:00 pm-3:00 am). Several IOP curve-derived parameters differed between the two groups, with larger amplitude during sleep time (P < 0.000) and longer duration of IOP ≥ 21 mmHg at awake time (P = 0.004) in the DON group than the OAG group. However, the diagnostic model generated from IOP parameters showed poor reliability (P = 0.001) in calibration test and was rejected. The other model built on PC factors achieved good performance of discrimination (AUC = 0.943) and calibration (P = 0.139) with a sensitivity of 87.50% and a specificity of 95.83% at cutoff value of 0.538 to identify OAG cases. CONCLUSION: The diagnostic model facilitates discrimination between OAG and DON in TED patients based on 24-h IOP-related patterns. TRIAL REGISTRATION: This work was registered on Chinese Clinical Trial Registry (ChiCTR1900025394).

VIP-HL: Semi-automated ACMG/AMP variant interpretation platform for genetic hearing loss.

The American College of Medical Genetics and Genomics, and the Association for Molecular Pathology (ACMG/AMP) have proposed a set of evidence-based guidelines to support sequence variant interpretation. The ClinGen hearing loss expert panel (HL-EP) introduced further specifications into the ACMG/AMP framework for genetic hearing loss. This study developed a tool named Variant Interpretation Platform for genetic Hearing Loss (VIP-HL), aiming to semi-automate the HL ACMG/AMP rules. VIP-HL aggregates information from external databases to automate 13 out of 24 ACMG/AMP rules specified by HL-EP, namely PVS1, PS1, PM1, PM2, PM4, PM5, PP3, BA1, BS1, BS2, BP3, BP4, and BP7. We benchmarked VIP-HL using 50 variants in which 82 rules were activated by the ClinGen HL-EP. VIP-HL concordantly activated 93% (76/82) rules, significantly higher than that of by InterVar (48%; 39/82). VIP-HL is an integrated online tool for reliable automated variant classification in hearing loss genes. It assists curators in variant interpretation and provides a platform for users to share classifications with each other. VIP-HL is available with a user-friendly web interface at http://hearing.genetics.bgi.com/.

AutoCNV: a semiautomatic CNV interpretation system based on the 2019 ACMG/ClinGen Technical Standards for CNVs.

BACKGROUND: The American College of Medical Genetics and Genomics (ACMG) and the Clinical Genome Resource (ClinGen) presented technical standards for interpretation and reporting of constitutional copy-number variants in 2019 (the standards). Although ClinGen developed a web-based CNV classification calculator based on scoring metrics, it can only track and tally points that have been assigned based on observed evidence. Here, we developed AutoCNV (a semiautomatic automated CNV interpretation system) based on the standards, which can automatically generate predictions on 18 and 16 criteria for copy number loss and gain, respectively. RESULTS: We assessed the performance of AutoCNV using 72 CNVs evaluated by external independent reviewers and 20 illustrative case examples. Using AutoCNV, it showed that 100 % (72/72) and 95 % (19/20) of CNVs were consistent with the reviewers' and ClinGen-verified classifications, respectively. AutoCNV only required an average of less than 5 milliseconds to obtain the result for one CNV with automated scoring. We also applied AutoCNV for the interpretation of CNVs from the ClinVar database and the dbVar database. We also developed a web-based version of AutoCNV (wAutoCNV). CONCLUSIONS: AutoCNV may serve to assist users in conducting in-depth CNV interpretation, to accelerate and facilitate the interpretation process of CNVs and to improve the consistency and reliability of CNV interpretation.

Telomerase activation by genomic rearrangements in high-risk neuroblastoma.

Neuroblastoma is a malignant paediatric tumour of the sympathetic nervous system. Roughly half of these tumours regress spontaneously or are cured by limited therapy. By contrast, high-risk neuroblastomas have an unfavourable clinical course despite intensive multimodal treatment, and their molecular basis has remained largely elusive. Here we have performed whole-genome sequencing of 56 neuroblastomas (high-risk, n = 39; low-risk, n = 17) and discovered recurrent genomic rearrangements affecting a chromosomal region at 5p15.33 proximal of the telomerase reverse transcriptase gene (TERT). These rearrangements occurred only in high-risk neuroblastomas (12/39, 31%) in a mutually exclusive fashion with MYCN amplifications and ATRX mutations, which are known genetic events in this tumour type. In an extended case series (n = 217), TERT rearrangements defined a subgroup of high-risk tumours with particularly poor outcome. Despite a large structural diversity of these rearrangements, they all induced massive transcriptional upregulation of TERT. In the remaining high-risk tumours, TERT expression was also elevated in MYCN-amplified tumours, whereas alternative lengthening of telomeres was present in neuroblastomas without TERT or MYCN alterations, suggesting that telomere lengthening represents a central mechanism defining this subtype. The 5p15.33 rearrangements juxtapose the TERT coding sequence to strong enhancer elements, resulting in massive chromatin remodelling and DNA methylation of the affected region. Supporting a functional role of TERT, neuroblastoma cell lines bearing rearrangements or amplified MYCN exhibited both upregulated TERT expression and enzymatic telomerase activity. In summary, our findings show that remodelling of the genomic context abrogates transcriptional silencing of TERT in high-risk neuroblastoma and places telomerase activation in the centre of transformation in a large fraction of these tumours.

Predictive value of pretreatment PD-L1 expression in EGFR-mutant non-small cell lung cancer: a meta-analysis.

OBJECTIVE: To investigate the predictive value of programmed death-ligand 1 (PD-L1) expression in non-small cell lung cancer (NSCLC) patients treated with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs). METHODS: We conducted a systemic search of PubMed, EMBASE, and the Cochrane Library from 1 January 2000 to 30 August 2020, to identify related studies. We combined the hazard ratio (HR) and 95% confidence interval (CI) to assess the correlation of PD-L1 expression with progression-free survival (PFS) and overall survival (OS). We assessed the quality of the included studies by the Newcastle-Ottawa Scale (NOS). We performed subgroup analyses based on immunohistochemistry (IHC) scoring system, IHC antibodies, sample size, countries, and survival analysis mode. Sensitivity analysis and evaluation of publication bias were also performed. RESULTS: Twelve studies including 991 patients met the criteria. The mean NOS score was 7.42 ± 1.19. Patients with high PD-L1 expression was associated with poorer PFS (HR = 1.90; 95% CI = 1.16-3.10; P = 0.011), while there was no association between PD-L1 expression and OS (HR = 1.19; 95% CI = 0.99-1.43; P = 0.070). Subgroup analysis prompted IHC scoring systems, IHC antibodies, and sample size have important effects on heterogeneity. The pooled results were robust according to the sensitivity analysis. CONCLUSIONS: The result of this meta-analysis suggested that PD-L1 expression might be a predictive biomarker for EGFR-mutant non-small cell lung cancer treated with EGFR-TKIs.

Identification of Two Novel Thalassemia Variants, HBA1: c.263delA and HBA2: c.376dupC, in Chinese Individuals.

We detected a novel frameshift variant (HBA1: c.263delA) and - -SEA (Southeast Asian), deletion in a 28-year-old Chinese woman with α-thalassemia (α-thal). This novel variant (a single nucleotide deletion at nucleotide 263 of codon 87) was detected by targeted next generation sequencing (NSG), resulting in a stop codon at amino acid 102 in exon 2 of the HBA1 gene. We also identified a novel heterozygous insertion (HBA2: c.376dupC) in a 24-year-old Chinese woman through screening for thalassemia. These two novel variants have expanded the mutation spectrum of α-thal and it would be beneficial for carrier screening, genetic counseling and prenatal diagnosis (PND) of α-thal.

Report of Two Novel Thalassemia Variants, HBB: c.181delG and HBA1: c.121_126delAAGACC, in Chinese Individuals.

In this study, we report two novel thalassemia variants detected in Chinese individuals using targeted NGS technology. We detected a novel frameshift variant, HBB: c.181delG, in a 32-year-old Chinese individual. This novel variant [a single nucleotide deletion at nucleotide 181 of codon 60 (-G)], was detected by targeted next generation sequencing (NGS), resulting in a stop codon at codon 60 in exon 2 of the HBB gene. The impact of this novel variant was further analyzed by an in vitro model. We also identified a novel in-frame variant, HBA1: c.121_126delAAGACC [codons 40/41 (-AAGACC)], in another Chinese individual in this study. We named these two novel variants, HBB: c.181delG and HBA1: c.121_126delAAGACC according to the Human Genome Variation Society (HGVS), which were detected by the first author. These two novel variants have expanded the mutation spectrum of thalassemia and it would be beneficial for carrier screening, genetic counseling and prenatal diagnosis (PND) of thalassemia.

Detection of the HBB: c.393T>G Mutation in Two Patients with Hypochromic Microcytic Anemia.

A novel mutation, HBB: c.393T>G on the HBB gene, was detected in two hypochromic microcytic anemia patients from Yulin, in the Guangxi Province of the People's Republic of China (PRC), by next-generation sequencing (NGS). It is a nonsense mutation causing a stop codon at amino acid 131 in exon 3 of the HBB gene. It was found in a heterozygous state in two patients who both presented severe anemia during pregnancy and moderate anemia before pregnancy; Hb A2 levels were slightly increased (more than 4.0%) in both patients. It was also detected in the father of one of the patients. This mutation was pathogenic, and caused the dominant thalassemia-like phenotypes in the two patients.

AutoPVS1: An automatic classification tool for PVS1 interpretation of null variants.

Null variants are prevalent within the human genome, and their accurate interpretation is critical for clinical management. In 2018, the ClinGen Sequence Variant Interpretation (SVI) Working Group refined the only criterion with a very strong pathogenicity rating (PVS1). To streamline PVS1 interpretation, we have developed an automatic classification tool with a graphical user interface called AutoPVS1. The performance of AutoPVS1 was assessed using 56 variants manually curated by the ClinGen's SVI Working Group; it achieved an interpretation concordance of 93% (52/56). A further analysis of 28,586 putative loss-of-function variants by AutoPVS1 demonstrated that at least 27.7% of them do not reach a very strong strength level, 17.5% because of variant-specific issues and 10.2% due to disease mechanism considerations. Notably, 41.0% (1,936/4,717) of splicing variants were assigned a decreased preliminary PVS1 strength level, a significantly greater fraction than in frameshift variants (13.2%) and nonsense variants (10.8%). Our results reinforce the necessity of considering variant-specific issues and disease mechanisms in variant interpretation and demonstrate that AutoPVS1 meets an urgent need by enabling biocurators to easily assign accurate, reliable and reproducible PVS1 strength levels in the process of variant interpretation. AutoPVS1 is publicly available at http://autopvs1.genetics.bgi.com/.

Concurrent hearing and genetic screening in a general newborn population.

Newborn hearing screening is not designed to detect delayed-onset prelingual hearing loss or aminoglycoside-antibiotic-induced ototoxicity. Cases with severe to profound hearing loss have been reported to have been missed by newborn hearing screens. The aim of this study was to evaluate the efficacy of concurrent hearing and genetic screening in the general population and demonstrate its benefits in practice. Enrolled newborns received concurrent hearing and genetic screens between September 1, 2015 and January 31, 2018. Of the 239,636 eligible infants (median age, 19 months), 548 (0.23%) had prelingual hearing loss. Genetic screening identified 14 hearing loss patients with positive genotypes and 27 patients with inconclusive genotypes who had passed the hearing screens. In addition, the genetic screen identified 0.23% (570/239,636) of the newborns and their family members as at-risk for ototoxicity, which is undetectable by hearing screens. In conclusion, genetic screening complements newborn hearing screening by improving the detection of infants at risk of hereditary hearing loss and ototoxicity, and by informing genotype-based clinical management for affected infants and their family members. Our findings suggest that the practice should be further validated in other populations and rigorous cost-effectiveness analyses are warranted.

Noninvasive prenatal diagnosis of cobalamin C (cblC) deficiency through target region sequencing of cell-free DNA in maternal plasma.

OBJECTIVE: This study aimed to validate the feasibility of haplotype-based noninvasive prenatal diagnosis (NIPD) of cobalamin C (cblC) deficiency. METHOD: This method includes three steps: First, targeted sequencing was performed on 21 families affected by cblC deficiency (including the couples and probands). Second, parental haplotypes linked with the pathogenic variant were determined using the genotypes of trios. Then, the fetal haplotypes were inferred through a parental haplotype assisted hidden Markov model (HMM). The NIPD results were confirmed by using the invasive procedures. RESULTS: Twenty-one fetal genotypes were successfully inferred by NIPD including three compound heterozygotes with cblC deficiency, nine heterozygote carriers of cblC deficiency, and nine normal fetuses. The NIPD results were confirmed using the invasive procedures with 100% concordant rate. CONCLUSION: This result has shown that haplotype-based NIPD of cblC deficiency has high concordant rate and indicated potential clinical utility as a pregnancy diagnosis method for high-risk carrier couples.

Noninvasive prenatal diagnosis for Duchenne muscular dystrophy based on the direct haplotype phasing.

OBJECTIVE: We aimed to investigate the validity of noninvasive prenatal diagnosis (NIPD) based on direct haplotype phasing without the proband or other family members and its feasibility for clinical application in the case of Duchenne muscular dystrophy (DMD). METHODS: Thirteen singleton-pregnancy families affected by DMD were recruited. The pathogenic variants in the pregnant females have been identified by multiplex ligation-dependent probe amplification (MLPA). We resolved maternal haplotypes for each family by performing targeted linked-read sequencing of their high molecular weight DNA, respectively. Then, we integrated the maternal haplotypes and the targeted sequencing results of maternal plasma DNA to infer the fetal haplotype and the DMD gene variant status. The fetal genotypes were further validated by using chorionic villus sampling. RESULTS: The method of directly resolving maternal haplotype through targeted linked-read sequencing was smoothly performed in 12 participated families, but one failed (F11). The predicted variant status of 12 fetuses was correct, which had been confirmed by invasive prenatal diagnosis. CONCLUSION: Direct haplotyping of NIPD based on linked-read sequencing for DMD is accurate.

Learning curve and clinical outcomes of percutaneous endoscopic transforaminal decompression for lumbar spinal stenosis.

PURPOSE: To define and analyze the learning curve of percutaneous endoscopic transforaminal decompression (PETD) for lumbar spinal stenosis (LSS). METHODS: From July 2015 to September 2016, 78 patients underwent PETD; one of whom was converted to open surgery, two were lost, and 75 were included in this study. Clinical results were assessed by using the Oswestry Disability Index (ODI) and visual analog scale (VAS). The learning curve was assessed by a logarithmic curve-fitting regression analysis. Of these 75 patients, 35 were defined as the "early" group, and 40 were defined as the "late" group for comparison. RESULTS: The mean follow-up was 25.37 ± 4.71 months. The median operative time gradually decreased from 95 (interquartile range, IQR, 85-110) minutes for the early group to 70 (IQR, 60-80) minutes for the late group (P < .000), and an asymptote was reached after approximately 35 cases. After surgery, the VAS for leg pain (LP) and ODI decreased significantly and remained constant during the follow-up. However, the VAS of low back pain (LBP) increased mildly. The total complication rate was 6.6%. ODI, VAS of LP and of LBP, and complication rate did not significantly differ between two groups. Early ambulation and short hospital stay after surgery were achieved. CONCLUSION: The learning curve of PETD for LSS was assessed and good clinical results were achieved. The surgeon's experience with this technique correlated with reduced operation time. Proper patient selection, familiarity with pathological anatomy, and manipulation under endoscopic view may shorten the learning curve and decrease complications.

[A consensus on the standardization of the next generation sequencing process for the diagnosis of genetic diseases (1) - Procedures prior to genetic testing].

Pre-testing preparation is the basis and starting point of genetic testing. The process includes collection of clinical information, formulation of testing scheme, genetic counseling before testing, and completion of informed consent and testing authorization. To effectively identify genetic diseases in clinics can greatly improve the diagnostic rate of next generation sequencing (NGS), thereby reducing medical cost and improving clinical efficacy. The analysis of NGS results relies, to a large extent, on the understanding of genotype-phenotype correlations, therefore it is particularly important to collect and evaluate clinical phenotypes and describe them in uniform standard terms. Different types of genetic diseases or mutations may require specific testing techniques, which can yield twice the result with half the effort. Pre-testing genetic counseling can help patients and their families to understand the significance of relevant genetic testing, formulate individualized testing strategies, and lay a foundation for follow-up.

[A consensus on the standardization of the next generation sequencing process for the diagnosis of genetic diseases (2) - Sample collection, processing and detection].

With high accuracy and precision, next generation sequencing (NGS) has provided a powerful tool for clinical testing of genetic diseases. To follow a standardized experimental procedure is the prerequisite to obtain stable, reliable, and effective NGS data for the assistance of diagnosis and/or screening of genetic diseases. At a conference of genetic testing industry held in Shanghai, May 2019, physicians engaged in the diagnosis and treatment of genetic diseases, experts engaged in clinical laboratory testing of genetic diseases and experts from third-party genetic testing companies have fully discussed the standardization of NGS procedures for the testing of genetic diseases. Experts from different backgrounds have provided opinions for the operation and implementation of NGS testing procedures including sample collection, reception, preservation, library construction, sequencing and data quality control. Based on the discussion, a consensus on the standardization of the testing procedures in NGS laboratories is developed with the aim to standardize NGS testing and accelerate implementation of NGS in clinical settings across China.