Detection of Genomic Variations in BRCA1 and BRCA2 Genes by Long-Range PCR and Next-Generation Sequencing
2012; Elsevier BV; Volume: 14; Issue: 3 Linguagem: Inglês
10.1016/j.jmoldx.2012.01.013
ISSN1943-7811
AutoresImma Hernán, Emma Borràs, Miguel Dias, María José Gamundi, Begoña Mañé, Gemma Llort, José A. G. Agúndez, Miguel Blanca, Miguel Carballo,
Tópico(s)DNA Repair Mechanisms
ResumoAdvances in sequencing technologies, such as next-generation sequencing (NGS), represent an opportunity to perform genetic testing in a clinical scenario. In this study, we developed and tested a method for the detection of mutations in the large BRCA1 and BRCA2 tumor suppressor genes, using long-range PCR (LR-PCR) and NGS, in samples from individuals with a personal and/or family history of breast and/or ovarian cancer. Eleven LR-PCR fragments, between 3000 and 15,300 bp, containing all coding exons and flanking splice junctions of BRCA1 and BRCA2, were obtained from DNA samples of five individuals carrying mutations in either BRCA1 or BRCA2. Libraries for NGS were prepared using an enzymatic (Nextera technology) method. We analyzed five individual samples in parallel by NGS and obtained complete coverage of all LR-PCR fragments, with an average coding sequence depth for each nucleotide of >30 reads, running from ×7 (in exon 22 of BRCA1) to >×150. We detected and confirmed 100% of the mutations that predispose to the risk of cancer, together with other genomic variations in BRCA1 and BRCA2. Our approach demonstrates that genomic LR-PCR, together with NGS, using the GS Junior 454 System platform, is an effective method for patient sample analysis of BRCA1 and BRCA2 genes. In addition, this method could be performed in regular molecular genetics laboratories. Advances in sequencing technologies, such as next-generation sequencing (NGS), represent an opportunity to perform genetic testing in a clinical scenario. In this study, we developed and tested a method for the detection of mutations in the large BRCA1 and BRCA2 tumor suppressor genes, using long-range PCR (LR-PCR) and NGS, in samples from individuals with a personal and/or family history of breast and/or ovarian cancer. Eleven LR-PCR fragments, between 3000 and 15,300 bp, containing all coding exons and flanking splice junctions of BRCA1 and BRCA2, were obtained from DNA samples of five individuals carrying mutations in either BRCA1 or BRCA2. Libraries for NGS were prepared using an enzymatic (Nextera technology) method. We analyzed five individual samples in parallel by NGS and obtained complete coverage of all LR-PCR fragments, with an average coding sequence depth for each nucleotide of >30 reads, running from ×7 (in exon 22 of BRCA1) to >×150. We detected and confirmed 100% of the mutations that predispose to the risk of cancer, together with other genomic variations in BRCA1 and BRCA2. Our approach demonstrates that genomic LR-PCR, together with NGS, using the GS Junior 454 System platform, is an effective method for patient sample analysis of BRCA1 and BRCA2 genes. In addition, this method could be performed in regular molecular genetics laboratories. Mutations that cause a loss of function in the tumor suppressor genes BRCA1 and BRCA2 predispose women to a high risk of breast and ovarian cancers.1King M.C. Marks J.H. Mandell J.B. New York Breast Cancer Study GroupBreast and ovarian cancer risks due to inherited mutations in BRCA1 and BRCA2.Science. 2003; 302: 643-646Crossref PubMed Scopus (1818) Google Scholar, 2Bordeleau L. Panchal S. Goodwin P. Prognosis of BRCA-associated breast cancer: a summary of evidence.Breast Cancer Res Treat. 2010; 119: 13-24Crossref PubMed Scopus (96) Google Scholar, 3Pruthi S. Gostout B.S. Lindor N.M. Identification and management of women with BRCA mutations or hereditary predisposition for breast and ovarian cancer.Mayo Clin Proc. 2010; 85: 1111-1120Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar Genetic testing for BRCA1 and BRCA2 mutations is a current practice for women with a family history of breast or ovarian cancer.4de Sanjosé S. Léoné M. Bérez V. Izquierdo A. Font R. 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Olopade O.I. Cummings S. Garber J.E. Bernhardt B. Antman K. Russo D. Wood M.E. Mullineau L. Isaacs C. Peshkin B. Buys S. Venne V. Rowley P.T. Loader S. Offit K. Robson M. Hampel H. Brener D. Winer E.P. Clark S. Weber B. Strong L.C. Thomas A. Sequence analysis of BRCA1 and BRCA2: correlation of mutations with family history and ovarian cancer risk.J Clin Oncol. 1998; 16: 2417-2425Crossref PubMed Scopus (415) Google Scholar BRCA1 and BRCA2 analysis to detect large exonic deletions or duplications that are not detectable by PCR amplification can be performed by the well-established multiplex ligation–dependent probe amplification technique14Bunyan D.J. Eccles D.M. Sillibourne J. Wilkins E. Thomas N.S. Shea-Simonds J. Duncan P.J. Curtis C.E. Robinson D.O. Harvey J.F. Cross N.C. Dosage analysis of cancer predisposition genes by multiplex ligation-dependent probe amplification.Br J Cancer. 2004; 91: 1155-1159Crossref PubMed Scopus (158) Google Scholar or by other methods.15Ewald I.P. Ribeiro P.L. Palmero E.I. Cossio S.L. Giugliani R. Ashton-Prolla P. Genomic rearrangements in BRCA1 and BRCA2: a literature review.Genet Mol Biol. 2009; 32: 437-446Crossref PubMed Scopus (70) Google Scholar However, BRCA1 and BRCA2 are large genes with many exons and, therefore, involve a considerable number of individual PCRs and sequencing reactions to cover the coding and flanking sequences of both genes. Although clinical protocols for genetic testing of BRCA1 and BRCA2 using PCR and Sanger sequencing are well established, the task is nevertheless costly and time-consuming. The introduction of massively parallel next-generation DNA sequencing (NGS) technology is becoming increasingly important in resequencing genes to characterize mutations that cause monogenic diseases.16Bowne S.J. Sullivan L.S. Koboldt D.C. Ding L. Fulton R. Abbott R.M. Sodergren E.J. Birch D.G. Wheaton D.H. Heckenlively J.R. Liu Q. Pierce E.A. Weinstock G.M. Daiger S.P. Identification of disease-causing mutations in autosomal dominant retinitis pigmentosa (adRP) using next-generation DNA sequencing.Invest Ophthalmol Vis Sci. 2011; 52: 494-503Crossref PubMed Scopus (74) Google Scholar, 17Kuhlenbäumer G. Hullmann J. Appenzeller S. Novel genomic techniques open new avenues in the analysis of monogenic disorders.Hum Mutat. 2011; 32: 144-151Crossref PubMed Scopus (91) Google Scholar Large platforms of NGS have been used for massively parallel DNA resequencing of multiple genes in pooled individuals.18ten Bosch J.R. Grody W.W. Keeping up with the next generation: massively parallel sequencing in clinical diagnostics.J Mol Diagn. 2008; 10: 484-492Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 19Voelkerding K.V. Dames S.A. Durtschi J.D. Next-generation sequencing: from basic research to diagnostics.Clin Chem. 2009; 55: 641-658Crossref PubMed Scopus (533) Google Scholar Genetically heterogeneous diseases frequently require the sequencing of many genes, sometimes also in many patients. Large next-generation platforms are indicated for this task. However, the cost and extremely large capacity of these platforms result in a loss of flexibility regarding the needs of many genetics laboratories, in which it is sometimes necessary to analyze (in a reasonably short time) samples from one or just a few individuals for mutations in large genes, such as BRCA1 and BRCA2. Recently, a scalable Roche 454 GS Junior bench top sequencing platform (Roche, Barcelona, Spain) has been introduced that is feasible for the sequencing of a subset of genes in individual samples using the NGS technique at assumable costs. By sharing the same core technology as the GS 20 and the GS FLX (Roche), the GS Junior combines single-molecule emulsion PCR with pyrosequencing.20Ronaghi M. Pyrosequencing sheds light on DNA sequencing.Genome Res. 2001; 11: 3-11Crossref PubMed Scopus (639) Google Scholar, 21Gharizadeh B. Herman Z.S. Eason R.G. Jejelowo O. Pourmand N. Large-scale pyrosequencing of synthetic DNA: a comparison with results from Sanger dideoxy sequencing.Electrophoresis. 2006; 27: 3042-3047Crossref PubMed Scopus (23) Google Scholar One approach for NGS resequencing involving many genes or large candidate genes, such as BRCA1 and BRCA2, to characterize the mutation that causes or may predispose to disease, requires the amplification of the exonic and flanking sequences by PCR as a previous step before sequencing. This involves the design and performance of many primers and PCRs to obtain complete coverage of the sequences of interest. Multiplex PCR is an attempt to approach this issue.22Varley K.E. Mitra R.D. Nested patch PCR enables highly multiplexed mutation discovery in candidate genes.Genome Res. 2008; 18: 1844-1850Crossref PubMed Scopus (49) Google Scholar However, multiplex PCR with many primer pairs often results in interpriming interactions or increases the mispriming events, thus preventing correct amplification.23Fan J.B. Chee M.S. Gunderson K.L. Highly parallel genomic assays.Nat Rev Genet. 2006; 7: 632-644Crossref PubMed Scopus (322) Google Scholar Therefore, a single PCR for each region of interest has to be performed, which is costly when hundreds of PCRs are required. A commercially available kit for BRCA1 and BRCA2 testing using a multiplex approach and NGS has been developed (Multiplicom N.V., Niel, Belgium). Recently, a novel multiplex open method for the 454 NGS platform of BRCA1 and BRCA2 has been reported.24De Leeneer K. Hellemans J. De Schrijver J. Baetens M. Poppe B. Van Criekinge W. De Paepe A. Coucke P. Claes K. Massive parallel amplicon sequencing of the breast cancer genes BRCA1 and BRCA2: opportunities, challenges, and limitations.Hum Mutat. 2011; 32: 335-344Crossref PubMed Scopus (58) Google Scholar Our goal was to develop a novel, simple, and effective method to detect DNA genomic variations in large genes, such as BRCA1 and BRCA2, which could be performed in normal genetics laboratories in hospitals. Most of these laboratories need to analyze a few patients for mutations in one or several genes associated with a genetic disease. This can become a costly and time-consuming task that is only feasible if many samples are processed together. However, except in a few large clinical institutions, this high concentration of patients is not usually found. The challenge is, therefore, to introduce rational methods of molecular analysis to study a few patients in a relatively short time and, thus, meet the clinical demand. We devised an effective approach using long-range PCR (LR-PCR) amplification and NGS to analyze the coding sequence (CDS) and flanking regions of the BRCA1 and BRCA2 genes. This approach could be extended to genetically heterogeneous diseases in which large or many genes are involved. Informed consent was obtained from all patients before the study, which was conducted in accordance with the Declaration of Helsinki and approved by the internal Clinical Research Ethics Committee of the Hospital of Terrassa, Terrassa, Spain. The DNA samples were from Spanish patients with a family history of breast or ovarian cancer, who had been previously screened for mutations in BRCA1 and BRCA2 coding exons and flanking regions by direct sequencing. DNA isolation from peripheral blood lymphocytes was performed automatically by the MagNaPure Compact Instrument (Roche) according to the manufacturer's protocol. Genomic reference sequences NG_005905.2 for BRCA1 and NG_012772.1 for BRCA2 were used to design the LR-PCR amplification. Pairs of primer sets were designed with the Oligo 7.41 (Molecular Biology Insights, Cascade, CO) program to amplify the complete CDS and flanking splice junctions of each gene. We took special care with the design of the primers to avoid the amplification of sequences of the BRCA1 pseudogene. Thus, for the LR-PCR fragment A, we amplified a DNA region of the BRCA1 gene that shares the 5′ region with the pseudogene but has a 3′ region that is absent in the pseudogene. To achieve this, we designed a forward primer in which the last 3′ nucleotide unmatched the pseudogene and the reverse primer hybridized specifically with the BRCA1 gene. Eleven pairs of primers (five for BRCA1 and six for BRCA2) were used to amplify the LR-PCR fragments. Amplification was performed in a 96-well Multiply-PCR plate (Sarstedt, Barcelona, Spain), in a preparation of 11 single PCRs, which renders DNA fragments between 3 and 16 kbp (Table 1). These fragments contain most of the genomic sequences of the genes analyzed. Each LR-PCR fragment sequence was verified once by Sanger sequencing.Table 1Amplified LR-PCR Fragments of BRCA1 and BRCA2FragmentGeneAmplified exonsLength (bp)PrimersAnnealing temperature (°C)ABRCA12–39436F: 5′-TGCCGGCAGGGATGTGCTTG-3′60.8R: 5′-TGCTTGCAGTTTGCTTTCACTGATGGA-3′BBRCA15–73081F: 5′-GTTTAGGTTTTTGCTTATGCAGCATCCA-3′61.8R: 5′-TCAGGTACCCTGACCTTCTCTGAAC-3′CBRCA18–104196F: 5′-GGAAAAGCACAGAACTGGCCAACA-3′60.9R: 5′-GTGGGTTGTAAAGGTCCCAAATGGT-3′DBRCA111–1312,739F: 5′-GCCAGTTGGTTGATTTCCACCTCCA-3′63.2R: 5′-TGCCTTGGGTCCCTCTGACTGG-3′EBRCA114–1913,853F: 5′-ACCCCCGACATGCAGAAGCTG-3′61.2R: 5′-GTGGTGCATTGATGGAAGGAAGCA-3′FBRCA120–2411,622F: 5′-TGACGTGTCTGCTCCACTTCCA-3′61R: 5′-AGTGAGAGGAGCTCCCAGGGC-3′GBRCA22–915,240F: 5′-CAGGCGGCGTTGGTCTCTAACTG-3′62.1R: 5′-AGGAGCAATCCTTCAATGGTGCC-3′HBRCA210–1315,294F: 5′-ACAGGAGAAGGGGTGACTGACCG-3′62.5R: 5′-GGGGAAAGCATCTCTGTTTGCTCT-3′IBRCA214–189043F: 5′-GCAAATGAGGGTCTGCAACAAAGGC-3′62.8R: 5′-TGAGAACAAGAGGGCAGCAAGC-3′JBRCA219–2411,617F: 5′-TGGTGGCTGAATAACCTTGGGCA-3′61.5R: 5′-TGGCATGGGAACAATGTGGCTT-3′KBRCA225–274382F: 5′-CCTGAGCTTTCGCCAAATTCAGCTAT-3′63R: 5′-TGCCCGATACACAAACGCTGAGG-3′F, forward; R, reverse. Open table in a new tab F, forward; R, reverse. Each reaction was performed in a 50-μL volume using the Expand Long Template PCR System (Roche) in buffer 3, with 500 μmol/L deoxyribonucleotide triphosphates, 300 nmol/L of each primer, and 3.75 U of enzyme. With a designed temperature gradient in the PCR annealing step, it is possible to amplify each DNA sample in a single row of a 96-well plate using a G-Strom gradient thermocycler (Ecogen, Barcelona, Spain). The PCR program consisted of the following: incubation at 94°C for 2 minutes; 10 cycles at 94°C for 10 seconds, gradient temperature annealing for 30 seconds, and 68°C for 13 minutes; followed by 20 cycles at 94°C for 15 seconds, gradient temperature annealing for 30 seconds, and 68°C for 13 minutes, with an increase of 20 seconds in each cycle. Finally, a temperature of 68°C was used for 7 minutes. The LR-PCR fragments were individually purified with the High Pure PCR Product Purification kit (Roche). They were then quantified using the Epoch Microplate Spectrophotometer combined with the Take3 Multi-Volume Plate (Izasa, Barcelona, Spain). The 11 purified fragments were then pooled to obtain an equal number of molecules of each LR-PCR fragment. The mix of the 11 LR-PCR DNA fragments that contained the complete coding and partial intronic sequences of the BRCA1 and BRCA2 genes was subjected to enzymatic fragmentation using Nextera (Ecogen, Barcelona, Spain) technology.25Caruccio N. Preparation of next-generation sequencing libraries using Nextera™ technology: simultaneous DNA fragmentation and adaptor tagging by in vitro transposition.Methods Mol Biol. 2011; 733: 241-255Crossref PubMed Scopus (59) Google Scholar This technology uses an in vitro transposition reaction that catalyzes the random nick of double-stranded breaks in the target DNA and covalently attaches the 3′ end of the transferred transposon strand to the 5′ end of the target DNA. Briefly, with incubation at 55°C for 5 minutes, 50 ng of pooled DNA was fragmented and tagged with the Nextera Enzyme Mix containing free transposon ends. The tagmented DNA was purified using a Zymo DNA Clean & Concentrator-5 Kit (Ecogen). Finally, the purified DNA was amplified by a limited-cycle PCR with a four-primer reaction to add Roche Titanium–compatible adaptor sequences. The primers used in this reaction were changed from the original ones provided by the Nextera vendor because we needed to add the compatible adaptor sequences to use in a GS Junior platform (Figure 1). Five DNA libraries, obtained in parallel from patients carrying a previously characterized mutation associated with an increased risk of breast or ovarian cancer, were performed by Nextera technology, as previously described. A sequence of 10 nucleotides, called the molecular identifier (MID), was added between the upstream sequence and the transposon end in the Nextera adaptors (Figure 1) to distinguish each sample after NGS. In this study, the MID 9 to 13 of the 454Standard MID set (Roche), compatible with a shotgun library protocol, was used. DNA libraries generated by Nextera technology need to be clonally amplified for sequencing in the 454 GS Junior platform. Accordingly, single-stranded template and emulsion PCRs were performed according to the emPCR Amplification Method Manual-Lib-A (Roche Applied Science). DNA sequencing was performed in a GS Junior NGS platform. Preparation of the sequencing run was performed as described in the Sequencing Method Manual (GS Junior Titanium Series; Roche Applied Science). We conducted three 454 sequencing runs. In one run, one sample was applied in a single sequencing plate (PicoTiter Plate). In the other two runs, equimolar quantities of the tagged fragments for the five samples were pooled together and a single sequencing reaction was performed. Thus, the five samples were loaded in the same PicoTiter Plate. We used Roche 454 GS Reference Mapper software version 2.5p1 to assemble and compare the 454 sequencing reads with the BRCA reference sequences (NG_005905.2 and NM_007294.3 for BRCA1 and NG_012772.1 and NM_000059.3 for BRCA2). Separate sequencing analysis was performed for each sample. The sequences of MID and transposon were filtered out before assembly and mapping. After signal processing for Shotgun, reads were mapped to the reference sequence and high-confidence differences (HCDiffs) were identified. The criteria for HCDiffs were defined by the GS Reference Mapper as variants detected in at least three nonduplicated reads of high quality in both forward and reverse reads and found in at least 10% of the total unique sequencing reads (nonduplicated, uniquely mapping reads that align at some location). We also used CLC Genomics Workbench version 4.8 (CLCBio, Arhus, Denmark) as an additional bioinformatics program. All sequence variants were named according to the Human Genome Variation Society recommendation guidelines, using the A of the ATG translation initiation codon as nucleotide +1. We classified each HCDiff as a single-nucleotide polymorphism (SNP) or a breast cancer–causing mutation. Both BRCA1 and BRCA2 genes have a large exon 11 that contains almost 50% of the CDS for BRCA1 and 60% for BRCA2. This exon 11 can be amplified by LR-PCR in a single fragment: D for BRCA1 and H for BRCA2. With 11 fragments (Table 1) of LR-PCR, we amplified all CDS of both genes. To generate long and accurate PCR fragments, a formulated blend of proofreading and high-fidelity polymerase was used (as described in Materials and Methods). We designed primers and conditions to allow the amplification of these genes in a simple robust PCR assay. The optimal annealing temperature for each fragment was determined, and amplification of the 11 DNA fragments was performed in a PCR program with a gradient of annealing temperatures. Thus, by using a 96-well plate, we could successfully amplify the 11 LR-PCR fragments for the five chosen samples in a single PCR run (Figure 2A). The LR-PCR products were analyzed by agarose gel electrophoresis (Figure 2B) and used for a library construction. By using Nextera technology, we obtained a library of BRCA1 and BRCA2 fragments from five different DNA samples ready to be sequenced in the 454 System. The samples selected for BRCA analysis by 454 sequencing had different types of breast cancer–causing mutations, including point mutations, small deletions, and small insertions (Table 2). We chose samples with these particular types of variants to evaluate the ability of the GS Junior sequencer to identify different types of sequence variation. Moreover, the samples carried several previously described SNPs. We sequenced the CDS and 20 bp (intronic flanking) of each exon of BRCA1 and BRCA2 in the five selected samples by the standard Sanger method. Variants detected by the 454 GS Junior platform were then compared with those obtained by Sanger sequencing. We analyzed the results from three GS Junior runs using GS Reference Mapper software version 2.5p1. One PicoTiter Plate contained only one sample, whereas the other two were replicates that contained five different samples that were distinguished with MIDs. We also analyzed the results in parallel with an external software program: CLC Genomics Workbench version 4.8 (CLCBio). The complete sequence analysis of BRCA1 and BRCA2 using this software showed a similar coverage profile to that obtained with GS Mapper software version 2.5p1 (Roche), but, in general, with a lower depth coverage; 99% of the validated variants were detected by both programs when default settings were used. However, CLC software detected more indels than Reference Mapper, although most of those found in poly(A) regions were false positive.Table 2BRCA Point Mutations Previously Identified in Each SampleSample no.GenePrimary mutation1BRCA2c.8851G>A2BRCA2c.3264dupT3BRCA2c.8174_8185delGGTATGCTGTTinsTT4BRCA1c.3359_3360delTT5BRCA1c.5123C>A Open table in a new tab In all of the sequencing runs, the GS Junior detected the mutations associated with breast cancer and the SNPs found with the Sanger method (Table 3, Table 4). The homozygous SNPs analyzed always gave total variation values >96.1% (mean, 99.0%), whereas the heterozygous SNPs showed values between a minimum of 33.3% and a maximum of 69.2% (mean, 47.8%).Table 3Mutations Identified in BRCA Genes by the 454 AssayGenePositionReference baseModified baseProtein changeNo. of samples/PTP51% VariantUnique depth% VariantUnique depthBRCA1c.3359_3360TT—⁎Denotes the deletion of TT bases.p.Val1120fs42.9210103BRCA1c.5123CAp.Ala1708Glu5450.5095BRCA2c.3264TTTp.Gln1089fs6034.546123BRCA2c.8174_8185GGTATGCTGTTATTp.Trp2725fs50.820.50105BRCA2c.8851GAp.Ala2951Thr46.324.50144PTP, PicoTiter Plate. Denotes the deletion of TT bases. Open table in a new tab Table 4The SNPs Identified in CDS of BRCA Genes by 454 AssayGeneVariantdb SNPNo. of samples/PTP15Sample 1Sample 2Sample 3Sample 4Sample 5% VariantUnique depth% VariantUnique depth% VariantUnique depth% VariantUnique depth% VariantUnique depth% VariantUnique depthBRCA1c.591C>T1799965014606307402603764.459BRCA1c.2077G>A498685040.213244.43634.46448.84104140.447BRCA1c.2082C>T179994943.812847.13433.36351.24104145.746BRCA1c.2311T>C1694042.511346.22633.34557.12803450.036BRCA1c.2612C>T79991741.613747.64246.86257.14204050.036BRCA1c.3113A>G1694138.114740.44757.77142.64704938.142BRCA1c.3548A>G1694243.410656.73053.85234.82303556.730BRCA1c.4308T>C106091540.66953.32140.63268.82203860.025BRCA1c.4837A>G179996667.95643.832682569.22603859.144BRCA2c.865A>C766173011143.330053035028034BRCA2c.1114A>C144848013703706104303454.951BRCA2c.1365A>G1801439012848.431062039036043BRCA2c.2229T>C180149901574030095051036062BRCA2c.2971A>G1799944014243.237065040029048BRCA2c.3396A>G180140696.112948.3291005743.24491.73439.543BRCA2c.4558A>G803586901001461003310078100371003310039BRCA2c.5744C>T4987117013851.24107604842.443060BRCA2c.6513G>C20607698.31731003198.77698.2571003110065BRCA2c.7242A>G17999559910453.1321004762.52410043035db SNP; PTP, PicoTiter Plate; SNP database (http://www.ncbi.nlm.nih.gov/snp, last accessed February 24, 2012). Open table in a new tab PTP, PicoTiter Plate. db SNP; PTP, PicoTiter Plate; SNP database (http://www.ncbi.nlm.nih.gov/snp, last accessed February 24, 2012). Libraries sequenced with GS Junior generated an average of 300-base sequence reads. An average of 8 × 104 high-quality reads (35% to 40% of total reads) per run was obtained. Of these reads, 38.5% and 33.3% were targeted in CDS for one- and five-sample runs, respectively. The average depths obtained in CDS of BRCA1 (RefSeq NM_007294.3) and BRCA2 (RefSeq NM_000059.3) were ×86 and ×109, respectively, when only one sample was analyzed; and ×30 and ×38, respectively, when five samples were analyzed together (Figure 3). However, analysis of total CDS depth showed some regions in BRCA1 with a low depth. These regions, comprising exons 10 (77 bp), 19 (41 bp), 20 (84 bp), 22 (74 bp), and 24 (125 bp), showed a significant low-depth coverage (<×10) in both one- and five-sample runs (see Supplemental Figure S1 at http://jmd.amjpathol.org). Graphic representation of the depth and coverage showed a similar profile for all samples in BRCA1 and BRCA2 sequencing runs, demonstrating the reproducibility of the method to prepare libraries for NGS. An analysis of depth in intronic regions showed some sequences that were overrepresented, whereas others were underrepresented. An analysis of these sequences (see Supplemental Figure S2 at http://jmd.amjpathol.org) illustrates that sequences of minor representation showed, in general, a significant content in homopolymeric A and T stretches. Moreover, these homopolymers were accurately sequenced up to seven nucleotides, but for longer sequences, inaccurate and inefficient sequencing was regularly observed. However, we also found poor coverage depth in regions free of homopolymeric stretches, suggesting that other unknown sequence effects of Nextera or sequencing specificity could be present. On the other hand, overrepresented sequences showed no more regions of higher melting stability or content in guanine and cytosine nucleotides than average. The results showed that variants detected by the GS Junior that could be considered false negative were always in regions containing poor sequence coverage (<×20 total depth) or had a total variation value <18%. In the coverage range of <20%, the variant validation as positive or false-negative change may be uncertain because, despite the fact that a few real heterozygous changes showed values near 50% variation, we also found variants with values around 30% that were not detected by Sanger, and were unlikely to be real changes. Thus, our results suggest a cutoff
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