Low-pass whole-genome sequencing in clinical cytogenetics: a validated approach

Transcription

1 Americn College of Medicl Genetics nd Genomics Originl Reserch Article Low-pss whole-genome sequencing in clinicl cytogenetics: vlidted pproch Zirui Dong, MSc 3, Jun Zhng, MD 4, Ping Hu, PhD 5, Hixio Chen, MPhil, Jinjin Xu, BSc, Qi Tin, MPhil 4, Lu Meng, MD 5, Ynchou Ye, BSc 4, Jun Wng, MPhil 6, Meiyn Zhng, BSc 6, Yun Li, MPhil 6, Huilin Wng, MPhil,3, Shnshn Yu, PhD, Fng Chen, MPhil,7, Jinsheng Xie, PhD, Hui Jing, PhD,9,, Wei Wng, PhD,6,8, Kwong Wi Choy, PhD,3 nd Zhengfeng Xu, MD 5 Purpose: Chromosoml microrry nlysis is the gold stndrd for copy-number vrint (CNV) detection in prentl nd postntl dignosis. We imed to determine whether next-genertion sequencing (NGS) technology could be n lterntive method for CNV detection in routine clinicl ppliction. Methods: Genome-wide CNV nlysis (>5 kb) ws performed on multicenter group of 57 ptients using low-coverge whole-genome sequencing pipeline. These smples were referred for chromosoml nlysis; CNVs (i.e., pthogenic CNVs, pcnvs) were clssified ccording to the Americn College of Medicl Genetics nd Genomics guidelines. Results: Overll, totl of 98 bortuses, 37 stillbirths, 49 prentl, nd 86 postntl smples were tested. Our pproch yielded results in 549 smples (96.3%). In ddition to 9 subjects with neuploidies, 3 pcnvs (74 losses nd 9 gins) were identified in 8 smples, giving dignostic yields of 53.% (95% confidence intervl: 45.8, 6.5), 4.7% (5., 3.), 8.5% (., 36.6), nd 3.% (3.6, 37.3) in ech group, respectively. Mosicism ws observed t level s low s 5%. Conclusions: Ptients with chromosoml diseses or microdeletion/ microdupliction syndromes were dignosed using high-resolution genome-wide method. Our study reveled the potentil of NGS to fcilitte genetic dignoses tht were not evident in the prentl nd postntl groups. Genet Med dvnce online publiction 8 Jnury 6 Key Words: moleculr kryotyping; next-genertion sequencing; pthogenic copy-number vrints INTRODUCTION DNA copy-number vrints (CNVs) ccount for up to 3 Mb of sequence vrition in norml humn individul, with vriety of lengths of up to millions of bse pirs., This represents the mjor genome diversity for two different individuls; some of these CNVs re known to be ssocited with the pthogenicity of vriety of humn disorders, including the commonly known DiGeorge syndrome (OMIM 884), 3 Angelmn syndrome (OMIM 583), 4 nd neurogenetic defects such s ATR syndrome (6p deletion; OMIM 6543, 6364, nd 63444). 5 To detect pthogenic CNVs, chromosoml microrry nlysis (CMA), including rry comprtive genomic hybridiztion (rry-cgh) 6,7 nd single-nucleotide polymorphism rry, 8,9 hs been widely used s gold stndrd. Compred with CMA, next-genertion sequencing (NGS) is n lterntive stte-of-the-rt technology promising improved detection of genetic bnormlities with unprecedented resolution. Recently, few retrospective studies with limited smple size hve supported the performnce of NGS for detecting CNVs in clinicl smples. 4 To study the dignostic effectiveness nd fesibility of using low-coverge (or low-pss) whole-genome NGS pproch to detect chromosoml numericl nd structurl bnormlities in dignostic lbortory, we pplied our in-house CNV detection method for multicenter group of 57 ptients referred to chromosoml nlysis. A totl of 98 bortuses, 37 stillbirths, 49 prentl smples, nd 86 postntl smples were tested. The first three uthors re co-first uthors. BGI-Shenzhen, Shenzhen, Chin; Deprtment of Obstetrics nd Gynecology, The Chinese University of Hong Kong, Hong Kong, Chin; 3 Shenzhen Reserch Institute, The Chinese University of Hong Kong, Shenzhen, Chin; 4 Deprtment of Obstetrics, The Third Affilited Hospitl of Sun Yt-sen University, Gungzhou, Chin; 5 Stte Key Lbortory of Reproductive Medicine, Deprtment of Prentl Dignosis, Nnjing Mternity nd Child Helth Cre Hospitl Affilited to Nnjing Medicl University, Nnjing, Chin; 6 Clinicl Lbortory of BGI Helth, BGI-Shenzhen, Shenzhen, Chin; 7 Shenzhen Birth Defect Screening Project Lb, BGI-Shenzhen, Shenzhen, Chin; 8 BGI-Nnjing, Nnjing, Chin; 9 The Gungdong Enterprise Key Lbortory of Humn Disese Genomics, BGI-Shenzhen, Shenzhen, Chin; Shenzhen Key Lbortory of Trnsomics Biotechnologies, BGI- Shenzhen, Shenzhen, Chin; Section of Moleculr Disese Biology, Deprtment of Veterinry Disese Biology, Fculty of Helth nd Medicl Sciences, University of Copenhgen, Copenhgen, Denmrk; Shenzhen Mternl nd Child Helth Cre Hospitl, Shenzhen, Chin. Correspondence: Kwong Wi Choy (richrdchoy@cuhk.edu.hk) nd Zhengfeng Xu (njxzf@6.com) nd Wei Wng (wngw@genomics.cn) Submitted July 5; ccepted 9 November 5; dvnce online publiction 8 Jnury 6. doi:.38/gim.5.99

2 Originl Reserch Article MATERIALS AND METHODS Subject enrollment nd smple recruitment The study ws pproved by the institutionl review bord of ech collbortive site. Written consent for storge nd subsequent nlysis ws obtined from ech prticipnt. Products of conception from first-trimester miscrrige nd fetl tissue from stillbirths were collected. For prentl smples, chorionic villi, mniotic fluid, nd cord blood were collected. A peripherl blood smple ws collected from ech postntl ptient who ws phenotypiclly bnorml nd referred for genetic testing. The YH lymphocytic cell line (the first Asin humn genome sequenced) 5 ws lso used for methodology evlution. Smple preprtion for NGS DNA ws extrcted with the use of commercil DNAextrction kit (Puregene; Qigen, Hilden, Germny) nd then quntified with the Qubit dsdna HS Assy Kit (Invitrogen, Life Technologies, Wlthm, MA) for DNA qulity control (QC) mesurement. All smples pssing QC (>5 ng; OD6/ OD8 >.8; OD6/OD3 >.5) were subsequently prepred for non-size-selected librry (~5 bp) protocol; in brief, ng of genomic DNA ws shered into smll frgments ( 3 bp) with Covris S (Covris, Woburn, MA). After end repir, ddition of n A overhng nd dpter ligtion, DNA frgments (without size selection) underwent cycles of polymerse chin rection (PCR). For the smples with size-selection librry construction, 5 ng of genomic DNA from ech smple ws first shered into smll (4 6 bp) frgments with Covris S. After end repir, ddition of n A overhng nd dpter ligtion, cycles of PCR were crried out using the DNA frgments with dpter molecules t both ends. The size-selection (55 65 bp) procedure for the PCR products ws performed vi % grose gel electrophoresis. Gel slices were excised nd purified with QIAquick Gel Extrction kit (Qigen) following the mnufcturer s protocol. PCR products from ech librry were subsequently purified with n Agencourt AMPure XP PCR Purifiction Kit (Beckmn Coulter, Bre, CA). The size distribution of ech librry ws ssessed using Bionlyzer DNA kit (Agilent Technologies, Snt Clr, CA). Concentrtions of the PCR products were mesured by quntittive PCR (qpcr). Non-size-selected librries with different index tgs with equl mollity were mixed into pool ( or 4 smples per lne) nd sequenced with 5-bse single-end sequencing (~5 million reds per smple) on HiSeq pltform (Illumin, Sn Diego, CA). Ech size-selected librry ws 5-bse pired end sequenced with ~9 million red pirs ( smples per lne), lso on the HiSeq pltform (Illumin). Red-depth estimtion, which is used to determine sufficient depth of coverge for trget region before further nlysis, ws performed for ech smple. It is clculted by multiplying the red mount produced (~5 million) nd the red length (5-bse single-end sequencing) nd dividing the result by the trget size (3 Gb s the size of the whole humn DONG et l Low-pss whole-genome sequencing in clinicl cytogenetics genome). Therefore, for ech smple with non-size-selected librry for CNV nlysis, red depth of ~.5 ws produced in this study. CNV nlysis Reds were ligned to the Ntionl Center for Biotechnology Informtion humn reference genome (hg9, GRCh37., herefter clled hg9) using SOAP (ref. 6), nd PCR duplictes were removed. Only uniquely mpped reds were selected. 3 Aprt from neuploidy dignosis 7 (Supplementry Methods online), in generl, CNV detection ws performed ccording to the three steps below:. Qulity control nd puttive CNVs screened with sliding windows. Mpped reds were clssified into djustble sliding windows, 8,9 which were 5 kb in length with 5 kb increments (Supplementry Methods nd Supplementry Figure S online), in terms of their mpped loctions (hg9). The coverge of ech window ws clculted by the red mount nd underwent two-step bis correction 3 (GC correction nd popultion-scle normliztion) (Supplementry Methods online). For the QC step, we first excluded the windows locted in the chromosomes of numericl disorders nd then clculted the genome-wide stndrd devition (GWSD) of the windows copy rtios. After two-step correction, the smple is considered to hve pssed QC if the GWSD is <.5, which is twice the GWSD of YH (Supplementry Figure S online). For smples tht pssed QC, cndidte CNV regions genome-wide were screened nd flgged for subsequent nlysis (Supplementry Methods online).. Precise boundries identified with increment rte of coverge of the djustble nonoverlpping windows. For more precise identifiction of CNV boundries (windows), the ligned reds were lso clssified into nonoverlpping windows. After tht, for ny prticulr djustble nonoverlpping window 8 (5 kb), the increment rtio of coverge (Figure nd Supplementry Figure S3 online) ws clculted s the coverge difference divided by its coverge. To detect the most precise boundries of ltered copy-number regions, we used circulr pek trough screening (Supplementry Methods nd Supplementry Figure S3 online) nd considered the verge copy number of the newly determined segment or region. 3. Individul CNV nnottion nd interprettion. A CNV is defined s deletion when its verge copy rtio is less thn.9 (mosics:.6 to.9) or s dupliction when greter thn. (mosics:. to.4), if this event is n outlier s determined by U-test (P vlue <.) from norml popultion with smples 3 (the Genomes Project). Clssifiction of CNVs is bsed on the Americn College of Medicl Genetics nd Genomics guidelines, with the criteri summrized below:

3 Low-pss whole-genome sequencing in clinicl cytogenetics DONG et l Originl Reserch Article. Pthogenic or likely pthogenic CNV: (i) contins pthogenic utosoml dominnt gene defined by GeneReviews; (ii) hrbors 5% of the length of criticl region of known syndrome defined in DECIPHER; (iii) covers the full length of pthogenic CNVs defined by ClinVrCNV or (iv) contins gene(s) reported in both OMIM nd HGMD. b. Vrint of uncertin significnce (VOUS, herefter referred to s CNV without further subclssifiction) is clssified s (i) covering the full length of VOUS defined by ClinVrCNV, or (ii) deleted nd contins gene(s) reported either in OMIM or HGMD only, or (iii) contins genes, but it is not known whether the genes in the intervl re dosgesensitive by CMA-bsed dtbses (ClinVrCNV, DECIPHER, nd in-house dtbses from Bylor College of Medicine nd The Chinese University of Hong Kong). CMA We used well-estblished customized 44K Fetl DNA Chip v. (Agilent Technologies) 7 nd Humn CytoSNP- BedChip with 3, probes (Illumin) for CMA. Single-nucleotide polymorphism rry nd rry-cgh tests were performed ccording to the mnufcturers protocols. For rry-cgh, CNVs were nlyzed vi CytoGenomics. 7 For single-nucleotide polymorphism rry, CNV nlysis ws crried out using KryoStudio V.3. nd GenomeStudio V. (ref. ) in prllel. Multiplex ligtion-dependent probe mplifiction vlidtion Probes (Supplementry Tble S4 online) were designed from the unique sequences within the CNV regions using RAW softwre (MRC-Hollnd BV, Amesterdm, the Netherlnds). Synthetic probes were diluted to finl concentrtion of 4 nmol/l, nd.5 µl ws dded to the P3 (MRC-Hollnd BV) Tble Chromosoml neuploidy nd CNV detection in the methodology vlidtion cohort of 7 smples NGS CMA Smple type Smple size C./Mos. pcnvs VOUS Benign b pcnvs VOUS Benign b Prentl 54 C. 43 c (3) c (3) 9 3 Mos. 4 (3) 4 (3) Postntl 7 C. 9 (9) (9) 8 Totl 7 Both 56 (4) (4) C., constitutionl CNV; CMA, chromosoml microrry nlysis; CNV, copy-number vrint; Mos., mosic CNVs; NGS, next-genertion sequencing; VOUS, vrint of uncertin significnce. pcnvs includes pthogenic nd likely pthogenic CNVs; numbers in prentheses indicte the number of smples detected to be crrying pcnvs. b Benign CNVs contins benign nd likely benign CNVs. c The number of pcnvs includes the trisomy 8 detected in smple DNJ67. X Y b X Y NGS Postntl 9 9 Prentl CMA Stillbirth Abortus Figure CNV detection in vlidtion group nd multicenter clinicl group. () The distribution of CNVs (including trisomy 8 detected in smple DNJ67) by CMA (inner circle) nd our NGS-bsed pproch (outer circle) in 7 vlidtion smples. Kryotypicl structures nd cytogenetic bnd colors re shown ccording to the University of Cliforni, Snt Cruz Genome Viewer Tble Browser nd chromosome color schemes (outmost circle). Rectngles in red nd blue indicte consistent copy-number losses nd gins, respectively, detected by both methods. Furthermore, for the CNVs uniquely clled by CMA (inner circle) or NGS (outer circle), losses nd gins re shown in purple nd in green, respectively. Three deletions (4q34.3q35., 5p5.33 nd 5q.), two duplictions (q5.q44 nd 7p3.3) nd tripliction (p.p3.33) in mosic fshion re shown (rrows) with mosic level (percentge). (b) The distribution of pthogenic or likely pthogenic CNVs detected in multicenter group including 98 erly bortuses, 37 stillbirths, 49 prentl smples, nd 86 postntl smples. Rectngles in red nd blue indicte the pthogenic copy-number losses nd gins, respectively. CMA, chromosoml microrry nlysis; CNV, copynumber vrint; NGS, next-genertion sequencing. 3

4 Originl Reserch Article probe mix. After hybridiztion, ligtion, mplifiction, nd electrophoresis with stndrd multiplex ligtion-dependent probe mplifiction procedure, dt were collected with 3 sequencer (ABI, Life Technologies) nd nlyzed with Gene Mrker.9 softwre (Softgenetics, Stte College, PA). Accession number Whole-genome sequencing reds re vilble in the Ntionl Center for Biotechnology Informtion Sequence Red Archive under ccession number SRA9678. Code vilbility All the progrms relevnt to this pipeline re vilble t sourceforge.net/projects/increment-rtio-of-coverge/files/ Increment_Rtio_of_Coverge.tr.gz/downlod. RESULTS To evlute the performnce of our in-house whole-genome low-coverge sequencing bsed pproch, we used DNA smples with known CMA results for comprison. Sixty-eight DNA smples (5 prentl nd 7 postntl smples) with vrious CNVs s well s 3 prentl smples with mosic pthogenic CNVs (pcnvs) were selected for NGS testing (~5 million reds or ~.5 per smple) in blinded fshion, nd the detection results were further compred with the CMA reports (Tble ). In ddition to identifying numericl bnormlities (Supplementry Tble S online), our pproch identified 4 constitutionl pcnvs rnging from.3 to 69. Mb in the prentl group (Tble, Supplementry Tble S online, nd Figure ), results tht were % consistent with the CMA reports. In the postntl group, our method identified nine constitutionl pcnvs rnging from.6 to.6 Mb, which were lso confirmed by CMA (Tble, Supplementry Tble S online, nd Figure ). For constitutionl pcnv detection, the NGS-bsed method provided.% (9.,.) sensitivity nd.% (89.,.) specificity. For mosic CNV detection, our NGS-bsed pproch detected six mosics, four of which were pcnvs (Figure nd Supplementry Tble S online), consistent with the three smples indicted with mosic pcnvs by CMA. In smple HKC637, there ws mosic copy-number gin of.9 Mb t bout the 4% level locted t 7p3.3( ) 3 (Figure,b nd Supplementry Tble S online) next to combintion of constitutionl deletion (67. kb) nd dupliction (57.5 kb). In smple HKC3, our pproch identified mosic deletion of 7. kb t 5p5.33( ) nd mosic tripliction of 34.8 Mb t p.p3.33( ) 3 4, both t bout the 5% level (Supplementry Tble S online). For the third smple, nmed HKC669, terminl mosic dupliction ws identified t q5.q44( ) 3, while terminl mosic deletion ws identified t 4q34.3q35.( ), both t bout the 5% level (Supplementry Tble S online). Overll, % DONG et l Low-pss whole-genome sequencing in clinicl cytogenetics concordnce between our NGS pipeline nd CMA ws chieved for chromosoml numeric nd pthogenic CNV detection, indicting tht the sensitivity nd specificity of pcnv (constitutionl nd mosic) detection were.% (9.4,.) nd.% (88.4,.), respectively, for our NGS-bsed pproch. We further selected 4 smples described bove with sufficient DNA for low-coverge pired-end sequencing to fine-mp the copy-number regions identified by our estblished method 3 (Supplementry Figure S4 online). Using simultion in the YH lymphocytic cell line 5 (Supplementry Tble S online) or nomlous red pirs detected by our blnced chromosoml rerrngement (or blnced chromosoml bnormlity) pipeline 3 (Supplementry Tble S3 online), we were ble to identify both the strt nd stop loctions of ech CNV within n djusted nonoverlpping window of ~5 kb from the precise region compred with n intervl of ~.5 kb by CMA (inner boundries, Supplementry Tble S3 online). Our pproch detected 3 dditionl CNV events s VOUS compred with CMA (Tble ), nd such dditionl informtion my in turn provide more importnt informtion for disese gene(s) discovery. For instnce, ptient ZS93 with DiGeorge syndrome (OMIM 884) 3 dignosed by clinicl phenotype ws defined s chromosomlly norml by CMA, but our pproch detected copy-number gin of 83.7 kb locted t 7q36.( ) 3 (Figure c) contining DPP6, overexpression of which hs been demonstrted to be ssocited with hert disese (i.e., fmilil idiopthic ventriculr fibrilltion). 4 Multiplex ligtion-dependent probe mplifiction vlidtion confirmed this copy-number gin (Figure d nd Supplementry Tble S4 online), which ws missed by CMA due to the probe design (Supplementry Figures S5 nd S6 online). The sme scenrio ws found in smple DNJ63 with congenitl hert disorder; in this ptient, 7.5 kb deletion ws detected t p.( ) hrboring gene MACROD. It is within n intron or involves some noncoding regions in some trnscripts of MACROD, nd hert disese is not consistent feture in DECIPHER cses with overlpping deletions in the region. However, muttion in this gene hs been reported to be ssocited with hert disese. 5 After evluting the sensitivity nd specificity of the NGS-bsed moleculr kryotyping in pcnv detection, we implemented this pltform in our routine dignostic lbortory setting to further evlute its performnce. We then obtined 57 smples from four tertiry referrl centers in Chin nd Hong Kong from Jnury 3 to Mrch 5. These smples included 98 bortuses, 37 stillbirths, 49 prentl smples, nd 86 postntl smples. Sequence-bsed nlysis ws successful in 549 smples (96.3%, Figure b). Among filed cses (/57), were bortuses from erly pregnncies, 3 were stillbirths, nd the other 8 were induced termintions with ultrsound nomlies. They ll hd poor DNA qulity, most likely due to fetl demise. Overll, we identified totl of,4 CNVs (79 losses nd,6 gins) in the 549 smples studied. Aneuploidies were identified in 9 smples, nd 4 pcnvs (74 losses nd 9 gins) were detected 4

5 Originl Reserch Article Low-pss whole-genome sequencing in clinicl cytogenetics DONG et l b C.Dup. (.54) Increment-rte C.Del. (.86) C.Dup. (.55) Mb. Mos.Dup. (.9, ~4%).5..5 C.Del. (.47). Mb 3. Mb Mos.Dup. (.,~4%) 4. Mb ,3 3,445 Loction (kb) c Increment-rte Cse 7q36.,, 8, 6, 4,, d.. Norml 7q ,4 53,5 53,6 Loction (kb) ,7 Are rtio:.553 f Norml Cse 7q36., 8, 6, 4,,.7.5 e 3,75 Cse Norml..5.. chr. chr. Figure Vlidtion of CNV detection by MLPA nd CMA. () Constitutionl deletion nd dupliction, followed by mosic copy-number gin detected by NGS in smple HKC637, which were confirmed by (b) CMA. C.Del., constitutionl deletion; C.Dup., constitutionl dupliction; Mos.Dup., mosic dupliction. Numbers in prentheses in indicte the copy rtio of the DNA frgment. Numbers in b show the logrtio provided by Agilent s Cytogenomics softwre. Percentge reflects the mosic level detected by our NGS-bsed pproch. (c) Identifiction of the precise region of dupliction locted in 7q36. ( ) 3 in smple ZS93. (d) MLPA vlidtion reflects the incresed dosge of 7q36.. (e) NGS nd (f) CMA results show consistent mosic trisomy (percentge: 4 5%) in smple 4S34. In nd c, blck lines indicte the distribution of copy rtios for djusted nonoverlpping windows (5 kb), nd ornge lines indicte the distribution of increment rte of coverge. In b nd f, dots in red, blue, nd blck indicte the copy-rtio loss, gin, nd eusomic, respectively. CMA, chromosoml microrry nlysis; CNV, copy-number vrint; MLPA, multiplex ligtion-dependent probe mplifiction; NGS, next-genertion sequencing. 5

6 Originl Reserch Article in 8 smples, giving n overll dignostic yield of 36.6% (3.6, 4.8; Tble ). In this group, NGS-bsed moleculr kryotyping identified cses of mosic neuploidies, of which the mjority were erly bortuses (/, Supplementry Tble S5 online); mternl cell contmintion ws excluded in ll cses by quntittive fluorescence polymerse chin rection. 6 Of the 549 successful cses, we rndomly selected 5 from 368 smples with sufficient DNA quntity (minimum 5 ng) for vlidtion using CMA. These smples included 5 neuploidies (3 mosics, Figure e,f nd Supplementry Tble S5 nd Supplementry Figure S7,b online) nd 3 pcnvs (Supplementry Tbles S6 S nd Supplementry Figure S7c,d online) within regions with probe coverge. All these events were % consistent with CMA, indicting high level of consistency nd robust performnce of the NGS pltform. Dignostic yield mong spontneous bortions nd stillbirths Chorionic villi or plcentl tissues from 98 bortuses, which were miscrried in the first trimester of pregnncy, were collected for DNA extrction nd subsequent sequencing. Ten smples filed QC (5.%, Supplementry Methods online). Of the 88 smples, 7 hd single neuploidy (38.3%); mong them, 9 smples hd sex-chromosome neuploidy, Supplementry Tble S5 online). In ddition, six smples (3.%) were dignosed with more thn one chromosome neuploidy (Supplementry Tble S5 online). Twelve smples (6.4%) were determined to hve 5 pcnvs (Supplementry Tble S6 online), rnging from kb to 78. Mb, nd 3 pcnvs were lrger thn Mb. A totl of smples were interpreted s utosoml neuploidy in mosic fshion (Supplementry Tble S5 online nd Figure c). The overll dignostic yield in the erly bortus group ws 53.% (/88, 45.8, 6.5) nd the incidence of chromosoml numericl bnormlities ws 46.8% (39.5, 54.). Fetl tissues (N = 35) nd mniotic fluids (N = ) from 37 stillbirths were studied. Three smples filed the QC check (8.%). Of the 34 smples, three (8.8%) with neuploidy (two smples with sex-chromosome neuploidy) were detected (Supplementry Tble S5 online). Two smples (5.9%) were found to hve three pcnvs (Supplementry Tble S7 online). The overll detection rte in this smple type ws 4.7% (5/34, 5., 3.). Dignostic yield in the prentl group Thirty-six mniotic fluid smples, 8 cord blood smples referred from high-risk pregnncies, nd 95 borted fetl smples (muscle, chorionic villus, or plcentl tissue) with ultrsound nomlies were recruited. Eight smples filed QC testing (5.4%). Of the 4 smples, (3.5%) hd numericl bnormlities, of which 6 were sex-chromosome neuploidies (Supplementry Tble S5 online). Nineteen smples (3.5%) were dignosed s hving 3 pcnvs (Supplementry Tble S8 online) rnging from 59.6 kb to 3.3 Mb, of which 7 were lrger thn Mb. For smples with prllel conventionl kryotyping, ll results DONG et l Low-pss whole-genome sequencing in clinicl cytogenetics were consistent with our NGS detection (Supplementry Tble S8 online). In ddition, smple 5B6364 ws identified s mosic for monosomy 3 (Supplementry Tble S5 online), but no culture smple ws vilble for confirmtion by fluorescence in situ hybridiztion confirmtion. In totl, the detection rte of chromosoml bnormlity in the prentl smple type ws 8.4% (4/4,., 36.6), nd, excluding neuploidies, the yield ws 3.5% (8.3,.3). Dignostic yield in the postntl group One hundred eighty-six whole-blood DNA smples were collected from individuls rnging from -dy-old newborns to dults with phenotypic nomlies, including couples (4 smples) requesting genetics dignosis nd counseling with history of pregnncy losses of fetuses with ultrsound nomlies. Of the 86 smples, 7 were identified s common trisomy syndromes (3.8%, Supplementry Tble S5 online). In 49 smples (6.3%, Supplementry Tble S9 online), 6 pcnvs were detected, rnging from 6.9 kb to 4.7 Mb. Three pirs of prentl DNAs were recruited for three probnds with pcnvs, nd two were dignosed s de novo (Supplementry Tble S9 online). The third smple, 3U553, ws suspected to be n unblnced segregnt of prentl blnced trnsloction becuse it hd deletion nd dupliction in the terminl regions of two chromosomes. To confirm this event, we pplied our blnced chromosoml bnormlity detection method to ech member from this trio nd identified chimeric red pirs supporting the probnd s kryotype s der(5) t(5;)(p4.;p3.)pt (Figure 3). This trnsloction ws lso confirmed by conventionl kryotyping (Figure 3b,c). In ddition, fmily member of smple 4B37388 with the sme phenotypic nomlies ws referred for testing. The probnd, 6-yer-old girl (4B37388), nd her 3-yer-old younger brother (4B593365) who presented with intellectul disbility nd speech dely, were found to hve common deletion in q3 (ref. 7), most likely due to n unblnced segregnt of prentl blnced trnsloction. Interestingly, smple 4B59339, phenotypiclly norml mle subject, displyed deletion of 3. kb (clssified s VOUS) in 6q6( )x tht contined PARK, which is relted to neurl development. 8 His wife hd three pregnncies with hydrocephlus; however, no DNA smples were obtined to confirm inheritnce to the hydrocephlic fetuses. In totl, the detection rte of chromosoml bnormlities in this postntl smple type ws 3.% (56/86, 3.6, 37.), providing n dditionl dignostic yield of.8% (9/49, 7.9, 9.3) mong kryotypiclly norml subjects. This is bsed on the ssumption tht pthogenic CNV size less thn 5 Mb could not be detected by conventionl cytogenetic nlysis. DISCUSSION This study ws designed to determine whether it would be more efficient to perform n NGS-bsed pproch rther thn conventionl CMA. In our vlidtion group, our NGS pproch ws % consistent with CMA nlysis for constitutionl nd 6

7 Low-pss whole-genome sequencing in clinicl cytogenetics DONG et l Originl Reserch Article Chr. b del () 4. Mb 4.5 Mb 3.5 Mb 4. Mb 4.5 Mb 5. Mb Chr. p3. p4. p4. Chr5 46,XY,t(5;)(p4.;p3.) GUCYC: NM_ Mb 3.3 Mb 3.34 Mb 3.36 Mb 3.38 Mb c -bp micro-deletion -bp micro-homology GUCYC: NM_4963 der(5)t(5;)(p4.;p3.)pt 3.8 Mb 3.3 Mb 4.78 Mb 4.8 Mb 4.8 Mb 4.84 Mb 4.86 Mb Chr5 p4.3 p3. p.3 Chr.. Mb.5 Mb 3. Mb 5. Mb 5.5 Mb 6. Mb Der() Chr. Figure 3 Composition of the derivtive chromosomes in blnced trnsloction detected by NGS nd confirmed by conventionl kryotyping. () Joining sequences of two derivtive chromosomes in pternl crrier. Precise brekpoints (shown in dotted line) re locted in p3. (4,78,9, bsed on hg9) nd 5p4. (3,34,435) with two bses of microdeletion (chrcters in red) nd microhomology (chrcters in blue), respectively, which were confirmed by Snger sequencing; (b nd c) conventionl G-bnded prtil kryotypes showing t(5;)(p4.;p3.) in fther nd der(5)t(5;)(p4.;p3.) pt in the probnd. CNV, copy-number vrint; NGS, next-genertion sequencing. Tble pcnv detection in multicenter cohort with four smple types Smple type Smples NGS successful Aneuploidy pcnvs Totl Erly bortus (95.%) 94 (88 c, 46.8% b, CI: 39.5, 54.) 5 ( c, 6.4% b, CI: 3.3,.9) 9 ( c, 53.% b, CI: 45.8, 6.5) Stillbirth (9.9%) 3 (3 c, 8.8% b, CI:.9, 3.7) 3 ( c, 5.9% b, CI:.7, 9.7) 6 (5 c, 4.7% b, CI: 5., 3.) Prentl 49 4 (94.6%) ( c, 4.9% b, CI: 9.5,.9) 3 (9 c, 3.5% b, CI: 8.3,.3) 44 (4 c, 8.4% b, CI:., 36.6) Postntl (.%) 7 (7 c, 3.8% b, CI:.6, 7.7) 6 (49 c, 6.3% b, CI:., 33.) 69 (56 c, 3.% b, CI: 3.6, 37.3) Totl (96.3%) 5 (9 c,.7% b, CI: 8.3, 5.4) 3 (8 c, 4.9% b, CI:., 8.) 9 ( c, 36.6% b, CI: 3.6, 4.8) CI, 95% confident intervl; NGS, next-genertion sequencing. pcnvs includes pthogenic nd likely pthogenic CNVs. b Percentge shows the frequency of the smples to be with neuploidy or pcnvs dividing by the number of smples with NGS successful yielded results in this smple type. c Number of smples detected to hve pcnvs in tht smple type. mosic pcnvs detection. Furthermore, 3 dditionl VOUS were detected becuse of more evenly distributed pproch (whole-genome sequencing nlysis) nd finer resolution of our lgorithm (5 kb) compred with probe selection by CMA. In our clinicl group, our NGS pproch reched dignostic yield of 36.6% (/ 549, 3.6, 4.8), indicting in.7 chromosoml numericl nd/or structurl nomlies mong those referred for genetic dignosis. This high detection rte ws due primrily to the much higher dignostic yield derived from the group of spontneous bortion thn tht from other smple types. However, this group in the current study is representtive group in clinicl dignostic lbs where chromosoml microdeletion/microdupliction nlysis hs been well pplied. For the stillbirth smples, owing to the smll smple size in this group (N = 34), smple bis my hve contributed to the higher dignostic yield tht ws observed, compred with recently published studies. 9,3 Nonetheless, our current dt truly show higher dignostic yield. For the prentl nd postntl smples, our NGS pproch gve much better dignostic yield thn conventionl kryotyping nlysis in tertiry referrl center. 7,3,3 Aprt from neuploidy detection, the prevlence of pthogenic or likely pthogenic CNVs ws 6.4% (3.3,.9), 5.9% (.7, 9.7), 3.5% (8.3,.3), nd 6.3% (., 33.) in erly bortuses, stillbirths, prentl smples, nd postntl smples, respectively. These re comprble to the dignostic yields detected by CMA, 7,9 3,33 35 indicting tht our detection rte ws consistent with the smple group/type. The medin size of pcnvs (N = 4) ws ~5. Mb, demonstrting the clinicl utility of NGS in moleculr dignostics. In ddition, bsed on CNV clssifiction s recommended by the Americn College of Medicl Genetics nd Genomics, 467 CNVs (64 losses nd 33 gins) were defined s VOUS 7

8 Originl Reserch Article rnging from 5. kb to.5 Mb (Supplementry Figure S8 online). Our NGS-bsed pproch identified,83 benign or likely benign CNVs in our clinicl groups (549 losses nd,74 gins) s defined by CMA-bsed dtbses. On verge, ech smple ws reported to crry 3.3 benign vrints (. loss nd.3 gins). Among them, 98.% were smller thn Mb, compred with 93.5% in the ClinVrCNV dtbse of benign CNVs. Overll, there ws significnt difference in CNV size between VOUS nd benign CNVs in this group (two-smple Kolmogorov-Smirnov test: P =.4, considered significnt t P <.5, Supplementry Figure S8 online). Therefore, comprehensive dtbse including dt generted from sequencing-bsed methods for studying the pthogenicity of VOUS will be useful. Our method employed popultion-bsed U-test for filtering out CNVs with high frequency in the control group or directly normlized by the control bseline. The reson tht there re still benign CNVs reported s VOUS is their low incidence (<5%) in norml popultions, requiring further clssifiction with CMA-bsed or even NGS-bsed dtbses. It is difficult to identify the phenotype of pcnv detected in n erly bortus, stillbirth, or prentl smple. In this scenrio, we cn use the postntl-bsed dtbse for interprettion of the clinicl significnce or prognosis. For instnce, prentl smple 4S647 ws referred for testing becuse of n ultrsound nomly identified t 3 weeks of gesttion. A deletion of.3 Mb locted t 5q3.q3.3( )x ws detected nd clssified s likely pthogenic becuse this deletion hs been implicted in the 5q3.3 deletion syndrome (OMIM 6), which hs been reported in postntl cses with typicl fetures (e.g., intellectul disbility nd seizures). 36 Becuse most well-known syndromes hve been chrcterized s hrboring one or more disese-cusing genes, identifiction of the precise boundries of copy-number chnges s detected by our NGS pltform is importnt for defining new syndrome. In this study, our pproch is demonstrted to be more precise in identifying the criticl region by our newly estblished increment rtio of coverge lgorithm. Compred to CMA, which is limited by probe spcing nd density, our NGS method is more ccurte nd precise for mpping the criticl region of diseses. For chromosoml rerrngement, nine smples (9/549) were noted to hve deletion nd dupliction in the terminl q/p rm of one or more chromosomes simultneously, implicting unblnced trnsloctions (Supplementry Tble S online). In NGS red-depth bsed methods, 37 only dosge chnges, rther thn the rel composition of derivtive chromosome(s), cn be observed. Therefore, kryotyping is recommended for investigtion of unblnced trnsloctions becuse of the sptil visuliztion. However, three smples hd terminl deletion or dupliction t submicroscopic level (<5 Mb), unlikely to be detectble by conventionl kryotyping but detectble by our estblished blnced chromosoml bnormlity detection method. 3 For prentl smples obtined from invsive testing, the turnround time is dys from smple receipt to dignostic report, which is competitive with CMA. Bsed on our dt using the HiSeq pltform, it is possible to evlute DONG et l Low-pss whole-genome sequencing in clinicl cytogenetics sequence informtion for up to 96 smples in single sequencing slide (two per run), which my ultimtely led to reducing the costs per ptient. Bsed on the regent costs including (i) DNA extrction (including DNA QC mesurement), (ii) librry construction (non-size-selected), nd (iii) bout 5 million single-end reds in the HiSeq pltform, costs re estimted t bout US$ per smple. Given stff cost s $5 per hour, s ech lne/run requires 6 working hours, the lbor cost is estimted to be $67 per smple (e.g., smples per lne). Therefore, in totl, the cost for ech smple would be bout $87, which compres fvorbly to the cost of conventionl kryotyping. In ddition, it llows more extensive numericl nd pcnv detection in vrious smple types, prticulrly in prentl dignosis, compred with the current stndrd of CMA. A limittion of this NGS-bsed CNV detection is, s with CMA, requirement for high-qulity DNA for testing. Therefore, this method my not be fully pplicble for DNA smples extrcted from fetl demise 9 (6.4%, /38 filed in our study). Moreover, neither rry-cgh nor low-coverge whole-genome sequencing with red-depth CNV-detection lgorithm cn detect triploidy. 38 In this study, n erly bortus smple (ID 4S697) ws identified by single-nucleotide polymorphism rry s 69,XXX but ws not detected by our ssy, which represents limittion of our method. Furthermore, triploid fetuses often result in erly spontneous bortions nd would hence go undetected. 38 With the exception of the bove limittions, our pproch provides high-throughput, robust, genome-wide high-resolution nlysis pipeline for detection of numericl disorders nd CNVs (prticulrly for pcnv). Compred with CMA, in the clinicl dignosis of neuploidy nd pcnvs, our NGS-bsed pproch shows equivlent effectiveness nd dvntges, including the detection of chromosoml mosicism t low level. In this study, smples displyed mosicism tht rnged from 5 to 7%. The NGS-bsed pproch hs been reported to detect lower-percentge mosics in other smple types (mternl plsm), 39 indicting the potentil to detect ccurtely lower levels of mosicism chromosoml bnormlities in our smple types. Second, our NGS pproch provides dditionl genome-wide detection of pcnvs or VOUS compred with CMA. In the vlidtion group, our pproch reveled 3 dditionl CNVs s VOUS, which my provide importnt informtion for gene discovery (Figure ). Finlly, the high success rte in our NGS-bsed pproch demonstrtes high dignostic rte in fetl demise 4 (93.6%), compred with 87.4% in microrry study on stillbirth. 9 In summry, chromosoml diseses or microdeletion/microdupliction syndromes cn be dignosed effectively by NGS. Our study demonstrted tht NGS is robust, sensitive, nd high-resolution genome-wide method to identify numericl nd pthogenic CNVs mong prentl nd postntl ptients. Furthermore, our study highlights the potentil for using NGS to fcilitte genetic dignoses in the prentl nd postntl smples tht hve not been detected by conventionl kryotyping nd/or CMA nlysis. 8

9 Low-pss whole-genome sequencing in clinicl cytogenetics DONG et l Originl Reserch Article SUPPLEMENTARY MATERIAL Supplementry mteril is linked to the online version of the pper t ACKNOWLEDGMENTS This study ws pproved by the Ntionl Bsic Reserch Progrm of Chin (CB9446), the Shenzhen Municipl Commission for Development nd Reform nd Key Lbortory Project in Shenzhen (CXB9366A nd CXB8596A), the Ntionl Nturl Science Foundtion of Chin (83495), the Medicl Leding Tlent nd Innovtion Tem Project of Jingsu Province (LJ9), nd the Key Technology R&D Progrm of Jingsu Province (BL39 nd F34). DISCLOSURE The uthors declre no conflict of interest. References. Abecsis GR, Auton A, Brooks LD, et l. An integrted mp of genetic vrition from,9 humn genomes. Nture ;49: Frzer KA, Murry SS, Schork NJ, Topol EJ. Humn genetic vrition nd its contribution to complex trits. Nt Rev Genet 9;: Jons RK, Montojo CA, Berden CE. The q. deletion syndrome s window into complex neuropsychitric disorders over the lifespn. Biol Psychitry 4;75: Greer PL, Hnym R, Bloodgood BL, et l. The Angelmn Syndrome protein Ube3A regultes synpse development by ubiquitinting rc. Cell ;4: Lmb J, Hrris PC, Wilkie AO, Wood WG, Duwerse JG, Higgs DR. De novo trunction of chromosome 6p nd heling with (TTAGGG)n in the lphthlssemi/mentl retrdtion syndrome (ATR-6). Am J Hum Genet 993;5: Tng YC, Amon A. Gene copy-number ltertions: cost-benefit nlysis. Cell 3;5: Leung TY, Vogel I, Lu TK, et l. Identifiction of submicroscopic chromosoml berrtions in fetuses with incresed nuchl trnslucency nd pprently norml kryotype. Ultrsound Obstet Gynecol ;38: Pinto D, Drvishi K, Shi X, et l. Comprehensive ssessment of rry-bsed pltforms nd clling lgorithms for detection of copy number vrints. Nt Biotechnol ;9: Gunderson KL, Steemers FJ, Lee G, Mendoz LG, Chee MS. A genome-wide sclble SNP genotyping ssy using microrry technology. Nt Genet 5;37: Mills RE, Wlter K, Stewrt C, et l.; Genomes Project. Mpping copy number vrition by popultion-scle genome sequencing. Nture ;47: Lui S, Song L, Crm DS, et l. Trditionl kryotyping vs copy number vrition sequencing for detection of chromosoml bnormlities ssocited with spontneous miscrrige. Ultrsound Obstet Gynecol 5;46: Ling D, Peng Y, Lv W, et l. Copy number vrition sequencing for comprehensive dignosis of chromosome disese syndromes. J Mol Dign 4;6: Li X, Chen S, Xie W, et l. PSCC: sensitive nd relible popultion-scle copy number vrition detection method bsed on low coverge sequencing. PLoS One 4;9:e Dun J, Zhng JG, Deng HW, Wng YP. Comprtive studies of copy number vrition detection methods for next-genertion sequencing technologies. PLoS One 3;8:e Wng J, Wng W, Li R, et l. The diploid genome sequence of n Asin individul. Nture 8;456: Li R, Yu C, Li Y, et l. SOAP: n improved ultrfst tool for short red lignment. Bioinformtics 9;5: Xie W, Tn Y, Li X, et l. Rpid detection of neuploidies on benchtop sequencing pltform. Prent Dign 3;33: Sztkiewicz JP, Wng W, Sullivn PF, Wng W, Sun W. Improving detection of copy-number vrition by simultneous bis correction nd red-depth segmenttion. Nucleic Acids Res 3;4: Li H, Run J, Durbin R. Mpping short DNA sequencing reds nd clling vrints using mpping qulity scores. Genome Res 8;8: Kerney HM, Thorlnd EC, Brown KK, Quintero-River F, South ST; Working Group of the Americn College of Medicl Genetics Lbortory Qulity Assurnce Committee. Americn College of Medicl Genetics stndrds nd guidelines for interprettion nd reporting of postntl constitutionl copy number vrints. Genet Med ;3: Hu P, Meng L, M D, et l. A novel p3 microdeletion encompssing PAX6 in Chinese Hn fmily with niridi, ptosis nd mentl retrdtion. Mol Cytogenet 5;8:3.. Eijk-Vn Os PG, Schouten JP. Multiplex ligtion-dependent probe mplifiction (MLPA ) for the detection of copy number vrition in genomic sequences. Methods Mol Biol ;688: Dong Z, Jing L, Yng C, et l. A robust pproch for blind detection of blnced chromosoml rerrngements with whole-genome low-coverge sequencing. Hum Mutt 4;35: Alders M, Koopmnn TT, Christins I, et l. Hplotype-shring nlysis implictes chromosome 7q36 hrboring DPP6 in fmilil idiopthic ventriculr fibrilltion. Am J Hum Genet 9;84: Slvin TP, Feng T, Schnell A, Zhu X, Elston RC. Two-mrker ssocition tests yield new disese ssocitions for coronry rtery disese nd hypertension. Hum Genet ;3: Cheng YK, Wong C, Wong HK, et l. The detection of mosicism by prentl BoBs. Prent Dign 3;33: Wilson HL, Wong AC, Shw SR, et l. Moleculr chrcteristion of the q3 deletion syndrome supports the role of hploinsufficiency of SHANK3/PROSAP in the mjor neurologicl symptoms. J Med Genet 3;4: Ling F, Li W, Zhng P, et l. A PARK polymorphism ssocited with delyed neuropsychologicl sequele fter crbon monoxide poisoning. BMC Med Genet 3;4: Reddy UM, Pge GP, Sde GR, et l.; NICHD Stillbirth Collbortive Reserch Network. Kryotype versus microrry testing for genetic bnormlities fter stillbirth. N Engl J Med ;367: Shlin E, Gustvsson P, Liedén A, et l. Moleculr nd cytogenetic nlysis in stillbirth: results from 48 consecutive cses. Fetl Dign Ther 4;36: Chong WW, Lo IF, Lm ST, et l. Performnce of chromosoml microrry for ptients with intellectul disbilities/developmentl dely, utism, nd multiple congenitl nomlies in Chinese cohort. Mol Cytogenet 4;7: Bruno DL, Gnesmoorthy D, Schoumns J, et l. Detection of cryptic pthogenic copy number vritions nd constitutionl loss of heterozygosity using high resolution SNP microrry nlysis in 7 ptients referred for cytogenetic nlysis nd impct on clinicl prctice. J Med Genet 9;46: Liu J, Bernier F, Luzon J, Lowry RB, Chernos J. Appliction of microrry-bsed comprtive genomic hybridiztion in prentl nd postntl settings: three cse reports. Genet Res Int ;: Lthi RB, Mssie JA, Loring M, et l. Informtics enhnced SNP microrry nlysis of 3 miscrrige smples compred to routine cytogenetics. PLoS One ;7:e Wpner RJ, Mrtin CL, Levy B, et l. Chromosoml microrry versus kryotyping for prentl dignosis. N Engl J Med ;367: Shrp AJ, Mefford HC, Li K, et l. A recurrent 5q3.3 microdeletion syndrome ssocited with mentl retrdtion nd seizures. Nt Genet 8;4: Zho M, Wng Q, Wng Q, Ji P, Zho Z. Computtionl tools for copy number vrition (CNV) detection using next-genertion sequencing dt: fetures nd perspectives. BMC Bioinformtics 3;4(suppl ):S. 38. McFdden DE, Robinson WP. Phenotype of triploid embryos. J Med Genet 6;43: Wllerstein R, Misr S, Dugr RB, Alem M, Mzzoni R, Grbedin MJ. Current knowledge of prentl dignosis of mosic utosoml trisomy in mniocytes: kryotype/phenotype correltions. Prent Dign 5;35: Grti FR, Gomes DM, Gnesmoorthy D, et l. Appliction of new moleculr technique for the genetic evlution of products of conception. Prent Dign 3;33:3 4. 9