Evaluation of human gene variant detection in amplicon pools by the GS-FLX parallel Pyrosequencer

Bordoni, Roberta; Bonnal, Raoul Jean Pierre; Rizzi, Ermanno; Carrera, Paola; Benedetti, Sara; Cremonesi, Laura; Stenirri, Stefania; Colombo, Alessio; Montrasio, Cristina; Bonalumi, Sara; Albertini, Alberto; Bernardi, L.; Ferrari, Maurizio; Bellis, Gianluca De

doi:10.1186/1471-2164-9-464

Cited by 18 publications

(16 citation statements)

References 23 publications

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“…With coverage below 2,000 times any conclusion about the mutant allele frequency is hard to make. In a recent study it was also shown that the variability of the values for the mutant allele fraction for heterozygote mutations decreased with increasing coverage [Bordoni et al, 2008]. We have shown that the confidence in mutation detection and frequency determination improves with higher coverage (less background).…”

Section: Discussionmentioning

confidence: 76%

“…The reason for these elevated rates could be that both of these mutations generate mutant alleles which consists of four consecutive A nucleotides. It has been shown that the Genome Sequencer FLX encounters difficulties when sequencing homopolymeric regions of more than 3 basepairs [Bordoni et al, 2008]. The Genome Sequencer FLX base-calling software calibrates the light emitted by single nucleotide incorporation, resulting in undercalls or overcalls in these positions, giving rise to homopolymer regions containing an incorrect number of nucleotides.…”

Section: Discussionmentioning

confidence: 99%

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Parallel sequencing used in detection of mosaic mutations: Comparison with four diagnostic DNA screening techniques

et al. 2009

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We have made an evaluation of mutation detection techniques for their abilities to detect mosaic mutations. In this study, Sanger sequencing, single-strand conformation polymorphism (SSCP)/heteroduplex analysis (HD), protein truncation test (PTT), and denaturating high-performance liquid chromatography (DHPLC) were compared with parallel sequencing. In total DNA samples from nine patients were included in this study. Mosaic mutations were artificially constructed from seven of these samples, which were from heterozygote mutation carriers with the mutant allele present at 50%. The mutations analyzed were as follows: c.646C>T, c.2626C>T, c.2828C>A, c.1817_1818insA, c.2788dupA, c.416_419delAAGA, and c.607delC in the APC gene. The lowest degree of mutant alleles detected with SSCP/HD and DHPLC varied between 5% and 25%, and between 15% and 50% for Sanger sequencing. Three of the mutations were analyzed with PTT with considerable variations in detection levels (from 10 to 100%). Using parallel sequencing a detection frequency down to 1% was reached, but to achieve this high sensitivity sufficient coverage was required. Two patients with natural mosaic mutations were also included in this study. These two mutations had previously been identified with Sanger sequencing (NF2 c.1026_1027delGA) and SSCP/HD (APC c.2700_2701delTC). In conclusion, all the evaluated methods are applicable for mosaic mutation screening even though combinations of the conventional methods should be used to reach an adequate sensitivity. Sanger sequencing alone is not sensitive enough to detect low mosaic levels. Parallel sequencing seems to be the ultimate choice but the possibilities to use this technique is today limited by its complexity, economics, and availability of instruments.

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Section: Discussionmentioning

confidence: 76%

Section: Discussionmentioning

confidence: 99%

Parallel sequencing used in detection of mosaic mutations: Comparison with four diagnostic DNA screening techniques

et al. 2009

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“…The findings obtained here have convinced us of the necessity of adopting a broader methodology, for example, 454 Roche sequencing (Ronaghi et al, 1998;Margulies et al, 2005;Bordoni et al, 2008) or ''whole-exome sequencing'' (Ng et al, 2010), to carry out a more extensive investigation on a larger group of genes and patients. Sequencing data will be filtered and the selected variations will be validated with a custom chip (GoldenGate, Illumina, San Diego, CA) (Lynch et al, 2009).…”

Section: Discussionmentioning

confidence: 81%

Unexpectedly low mutation rates in beta‐myosin heavy chain and cardiac myosin binding protein genes in italian patients with hypertrophic cardiomyopathy

Roncarati

Latronico

Musumeci

et al. 2011

Journal Cellular Physiology

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Hypertrophic cardiomyopathy (HCM) is the most common genetic cardiac disease. Fourteen sarcomeric and sarcomere-related genes have been implicated in HCM etiology, those encoding β-myosin heavy chain (MYH7) and cardiac myosin binding protein C (MYBPC3) reported as the most frequently mutated: in fact, these account for around 50% of all cases related to sarcomeric gene mutations, which are collectively responsible for approximately 70% of all HCM cases. Here, we used denaturing high-performance liquid chromatography followed by bidirectional sequencing to screen the coding regions of MYH7 and MYBPC3 in a cohort (n = 125) of Italian patients presenting with HCM. We found 6 MHY7 mutations in 9/125 patients and 18 MYBPC3 mutations in 19/125 patients. Of the three novel MYH7 mutations found, two were missense, and one was a silent mutation; of the eight novel MYBPC3 mutations, one was a substitution, three were stop codons, and four were missense mutations. Thus, our cohort of Italian HCM patients did not harbor the high frequency of mutations usually found in MYH7 and MYBPC3. This finding, coupled to the clinical diversity of our cohort, emphasizes the complexity of HCM and the need for more inclusive investigative approaches in order to fully understand the pathogenesis of this disease. J. Cell. Physiol. 226: 2894–2900, 2011. © 2011 Wiley-Liss, Inc.

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“…With respect to read percentage required for variant identification, we set our criterion to >35% to minimize false‐positives, in turn anticipating false‐negatives. It has been shown that the Genome Sequencer FLX system encounters difficulties when sequencing homopolymeric regions of more than 3 bp [Bordoni et al, 2008], and such stretches turned out to be major sources of sequencing errors. With the newly developed Titanium technology and software, containing various quality filters to remove poor‐quality sequence, longer strings of up to 6 bp could be resolved very well; one example is shown in this study.…”

Section: Discussionmentioning

confidence: 99%

Rapid and efficient human mutation detection using a bench-top next-generation DNA sequencer

et al. 2011

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Next-generation sequencing (NGS) technologies can be a boon to human mutation detection given their high throughput: consequently, many genes and samples may be simultaneously studied with high coverage for accurate detection of heterozygotes. In circumstances requiring the intensive study of a few genes, particularly in clinical applications, a rapid turn-around is another desirable goal. To this end, we assessed the performance of the bench-top 454 GS Junior platform as an optimized solution for mutation detection by amplicon sequencing of three type 3 semaphorin genes SEMA3A, SEMA3C and SEMA3D implicated in Hirschsprung disease (HSCR). We performed mutation detection on 39 PCR amplicons totaling 14,014bp in 47 samples studied in pools of 12 samples. Each 10-hour run was able to generate ∼75,000 reads and ∼28 million high-quality bases at an average read length of 371bp. The overall sequencing error was 0.26 changes per kb at a coverage depth of ≥20 reads. Altogether, 37 sequence variants were found in this study of which 10 were unique to HSCR patients. We identified five missense mutations in these three genes that may potentially be involved in the pathogenesis of HSCR and need to be studied in larger patient samples.

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Evaluation of human gene variant detection in amplicon pools by the GS-FLX parallel Pyrosequencer

Cited by 18 publications

References 23 publications

Parallel sequencing used in detection of mosaic mutations: Comparison with four diagnostic DNA screening techniques

Parallel sequencing used in detection of mosaic mutations: Comparison with four diagnostic DNA screening techniques

Unexpectedly low mutation rates in beta‐myosin heavy chain and cardiac myosin binding protein genes in italian patients with hypertrophic cardiomyopathy

Rapid and efficient human mutation detection using a bench-top next-generation DNA sequencer

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