Littérature scientifique sur le sujet « 2nd generation sequencing »
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Articles de revues sur le sujet "2nd generation sequencing"
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Gut, Ivo G. « 2nd generation DNA sequencing meets functional genomics ». Aging 2, no 9 (12 septembre 2010) : 541. http://dx.doi.org/10.18632/aging.100199.
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Mahmoud, Medhat, Marek Zywicki, Tomasz Twardowski et Wojciech M. Karlowski. « Efficiency of PacBio long read correction by 2nd generation Illumina sequencing ». Genomics 111, no 1 (janvier 2019) : 43–49. http://dx.doi.org/10.1016/j.ygeno.2017.12.011.
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Dagogo-Jack, Ibiayi, Marguerite Rooney, Rebecca Nagy, Subba Digumarthy, Emily Chin, Jennifer Ackil, Justin F. Gainor, Jessica Jiyeong Lin, Richard B. Lanman et Alice Tsang Shaw. « Longitudinal analysis of plasma ALK mutations during treatment with next-generation ALK inhibitors. » Journal of Clinical Oncology 37, no 15_suppl (20 mai 2019) : 9068. http://dx.doi.org/10.1200/jco.2019.37.15_suppl.9068.
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9068 Background: Next-generation ALK tyrosine kinase inhibitors (TKIs) are the cornerstone of management of ALK-positive (ALK+) lung cancer. Each ALK TKI has a unique spectrum of activity against distinct ALK kinase domain mutations (muts). Plasma genotyping is a promising strategy for identifying ALK muts at relapse on ALK TKIs. Methods: To detect ALK muts, we performed next-generation sequencing (Guardant360) of circulating tumor DNA from patients (pts) with ALK+ lung cancer relapsing on a second-generation (2nd-gen) ALK TKI (n = 65) or the third-generation (3rd-gen) TKI lorlatinib (n = 26). Results: Among 65 pts progressing on a 2nd-gen TKI, 49 (75%) had only received one 2nd-gen ALK TKI prior to analysis: n = 42 alectinib, n = 3 each brigatinib/ceritinib, and n = 1 ensartinib. We detected an ALK mut in 42/65 (65%) specimens at relapse, among which ALK G1202R (32%) and I1171X (23%) were the most common. Sixteen (25%) pts had ≥2 ALK muts at progression on a 2nd-gen TKI. Among 26 pts progressing on lorlatinib (all of whom had previously relapsed on a 2nd-gen ALK TKI), we identified ALK muts in 20 (76%), including 14 (54%) with ≥2 ALK muts. Detection of ≥2 ALK muts was more common at relapse on lorlatinib compared to a 2nd-gen TKI (p = 0.013). To assess the evolution of ALK muts during treatment with different TKIs, we analyzed serial plasma specimens from 20 pts treated with sequential 2nd-gen/2nd-gen or 2nd-gen/3rd-gen TKIs. Among six pts who received alectinib followed by brigatinib, repeat plasma analysis at brigatinib progression revealed persistence of pre-brigatinib ALK muts in two pts (one L1196M and one G1202R), expansion of G1202R in one pt, and acquisition of new ALK muts in three pts. Among 14 pts who received a 2nd-gen TKI followed by lorlatinib, 11 had persistence of pre-lorlatinib ALK muts and 8 acquired ≥1 additional ALK muts at lorlatinib progression. The most frequently acquired ALK mut was D1203N in four of eight cases. Conclusions: ALK resistance muts are prevalent at relapse on next-generation ALK TKIs and increase with each successive generation of ALK TKIs. These findings suggest that sequential therapy with increasingly potent ALK TKIs may select for compound ALK muts and/or fuel tumor heterogeneity.
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Roeper, Julia, Maria Netchaeva, Anne Christina Lueers, Ursula Stropiep, Cora Hallas, Markus Tiemann, Nicole Neemann et al. « Impact on OS of 2nd and 3rd generation TKI in EGFR mt+ and ALK+ patients : Results of the NOWEL network. » Journal of Clinical Oncology 35, no 15_suppl (20 mai 2017) : e20560-e20560. http://dx.doi.org/10.1200/jco.2017.35.15_suppl.e20560.
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e20560 Background: Available clinical research data shows that early mutation testing for patients with NSCLC stage IV could lead to an effective choice of therapy for patients with proven mutations. Targeted therapies achieve a higher ORR, PFS, OS and a better quality of life than chemotherapy in mt+ patients. With the advent of 2nd and 3rd generation TKI´s effective in 1st generation TKI resistant tumors, we wanted to study the impact of these drugs on the outcome of patients in a real life setting in 3 lung cancer centers. Methods: 1383 patients from the three cancer centers diagnosed with NSCLC stage IV (UICC 7) were examined. Methods for the detection of mutations included Sanger Sequencing, hybridization based COBAS testing as well as hybrid cage next generation sequencing. Results: 880/1383 (64%) consecutive patients with non-squamous cell NSCLC from the cancer centers were studied for the presence of tumor mutations, especially for EGFR and ALK mutations. The EGFR mutation rate was 16.6% (141/880), and the ALK-translocation rate 3.8% (24/635). Median OS in EGFR mt+ patients was 31 (n = 78) vs. 32 (n = 38) vs. 16 (n = 14) months respectively (center 1 vs. center 2 vs. center 3). Median OS in ALK mt+ patients was 25 (n = 17) months in center 1 and 11 (n = 5) months in center 2 (p < 0.05). Use of 3rd generation TKI Osimertinib (n = 17) lead to a significantly higher OS (n = 17, median OS 67 mo) than the use of only 1st and 2nd generation TKI (n = 113, median OS 24 mo, p < 0.000). Similarly, use of 2nd and 3rd generation ALKi impacted significantly on median OS: Crizotinib alone n = 7, 17 months, Crizotinib followed by Ceritinib and/or Brigatinib (n = 9) median OS not reached, p < 0.001. Conclusions: Smalldifferences in OS were observed, depending on the treatment centers, but the use of multiple EGFR and ALK-I impacted highly significantly on the outcome of patients with EGFR and ALK-alterations in a real life setting.
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Ji, Hezhao, Neil Parkin, Feng Gao, Thomas Denny, Cheryl Jennings, Paul Sandstrom et Rami Kantor. « External Quality Assessment Program for Next-Generation Sequencing-Based HIV Drug Resistance Testing : Logistical Considerations ». Viruses 12, no 5 (18 mai 2020) : 556. http://dx.doi.org/10.3390/v12050556.
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Next-generation sequencing (NGS) is likely to become the new standard method for HIV drug resistance (HIVDR) genotyping. Despite the significant advances in the development of wet-lab protocols and bioinformatic data processing pipelines, one often-missing critical component of an NGS HIVDR assay for clinical use is external quality assessment (EQA). EQA is essential for ensuring assay consistency and laboratory competency in performing routine biomedical assays, and the rollout of NGS HIVDR tests in clinical practice will require an EQA. In September 2019, the 2nd International Symposium on NGS HIVDR was held in Winnipeg, Canada. It convened a multidisciplinary panel of experts, including research scientists, clinicians, bioinformaticians, laboratory biologists, biostatisticians, and EQA experts. A themed discussion was conducted on EQA strategies towards such assays during the symposium. This article describes the logistical challenges identified and summarizes the opinions and recommendations derived from these discussions, which may inform the development of an inaugural EQA program for NGS HIVDR in the near future.
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Suryavanshi, Moushumi, Sakshi Mattoo, Sanjeev Kumar Sharma, Anurag Mehta et Ullas Batra. « Primary and secondary resistance mechanisms in first, second and third generation tyrosine kinase inhibitors in EGFR mutant non-small cell lung cancer patients. » Journal of Clinical Oncology 39, no 15_suppl (20 mai 2021) : e21142-e21142. http://dx.doi.org/10.1200/jco.2021.39.15_suppl.e21142.
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e21142 Background: Different molecular mechanisms of on target and off target primary and secondary resistance have been observed in EGFR mutant NSCLC patients after first(1st), second(2nd) and third (3rd) generation of tyrosine kinase inhibitors(TKIs). Next generation sequencing(NGS) offers a comprehensive method of detecting these mechanisms to decide next line of treatment. Methods: We retrospectively analyzed 430 samples of NSCLC for primary and secondary resistance to 1st, 2nd and 3rd TKIs. NGS was performed using thermofischer Ion Torrent Oncomine Focus 52 gene Assay. These cases were divided into 4 groups.1)Primary resistance to first and second generation TKIs 2)Primary resistance to 3rd generation TKI 3)Secondary resistance to 1st and 2nd generation TKI 4) Secondary resistance to 3rd generation TKI.Last group was further subgrouped into A when 3rd generation TKI was offered as second line after 1st or 2nd generation TKIs on detection of T790M and subgroup B when it was given as first line. Results: Group1 had 13 cases. There were 2 cases of complex EGFR exon 19 mutation p.Glu746_Leu747delinsValPro, 4 cases of EGFR exon 20 insertion, 1 case of dual EGFR L833V & H835L mutation, 2 cases with EGFR amplification with EGFR exon 19 del and PIK3CA C420_P421del along with EGFR exon 19 del. Four cases had no additional abnormality. Group 2 had 5 cases:1 case had L858R and E709A dual mutation, 2 cases had KRAS G13C and KRAS G12V along with EGFR exon 19 del. One case had EGFR amplification and one case had MET amplification along with EGFR exon 19 del respectively.Group 3 had 34 cases including 10 cases of EGFR L858R and 24 cases of exon 19 deletion.T790M mutation was detected in 8 patients, MET amplification in 7 cases,one case had both T790M and MET amplification. One case lost the primary EGFR exon 19 del. Others mutations detected were KRAS G13C, PIK3CA H1047R, TP53 R213Q and TP53 C242fs. Group3 had 15 cases with 7 cases in subgroup A and 9 cases in subgroup B. In subgroup A T790M mutation was lost in 6 out of 7 cases.One case which lost T790M developed ALK translocation.One case of EGFR exon 19 del retained EGFR T790M with EGFR C797S in cis allele. Other mutations detected were PIK3CA E542K and KRAS G12C. In subgroup B one case showed EGFR C797S(both cis and trans) besides the primary EGFR exon 19 del. One case showed BRAF G469A along with EGFR exon 19 del. Other mutations detected were CTNNB1 D32N, KRAS G12V, and PIK3CA E542K. Conclusions: Primary and secondary acquired resistance is unavoidable in EGFR mutant advanced NSCLC on any generation of TKIs. NGS offers an advantage in diagnosing mechanism of resistance for further choice of therapy.
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Tsai, Cheng-Hong, Jih-Luh Tang, Feng-Ming Tien, Yuan-Yeh Kuo, Chien-Chin Lin, Mei-Hsuan Tseng, Yen-Ling Peng et al. « Minimal Residual Disease Monitoring By Next-Generation Sequencing in Patients with Acute Myeloid Leukemia : MRD Positivity after First Consolidation Chemotherapy Can Better Predict Clinical Outcomes Than That after Induction Chemotherapy ». Blood 134, Supplement_1 (13 novembre 2019) : 2698. http://dx.doi.org/10.1182/blood-2019-126870.
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Introduction Presence of minimal residual disease (MRD) detected by multicolor flow cytometry (MCFC) or quantitative polymerase chain reaction has been recognized as an independent important prognosticator for patients with acute myeloid leukemia (AML). Next-generation sequencing (NGS) can simultaneously detect various mutations and be applied to the majority of patients with AML, but the clinical implication of its use in MRD monitoring remains to be clarified. Recently, it was shown that NGS MRD of mutants other than the common mutations occurring in clonal hematopoiesis of indeterminate potential, including the DTA (DNMT3A, TET2, and ASXL1) mutations, carry prognostic impacts on relapse rates and overall survival (OS) in AML patients. However, the proper time point for NGS MRD detection after treatment is still unclear. Our hypothesis is that the NGS MRD detected at different time points might have different clinical implications. In this regard, we aimed to explore the clinical implication of NGS MRD at different time points in AML patients after chemotherapy. Method We enrolled 306 de novo non-M3 and non-M6 AML patients who attained complete remission (CR) after standard induction chemotherapy and received 2-4 courses of post-remission chemotherapy with high-dose cytarabine with or without anthracycline. We analyzed bone marrow samples serially collected at diagnosis, first CR (1st time point for MRD analysis), and after the first consolidation chemotherapy (2nd time point). We used the TruSight myeloid panel (Illumina) to survey the 54 genes related to myeloid malignancies. Because of the sequencing sensitivity issue, we excluded CEBPA mutation and FLT3-ITD in the subsequent analyses. The median follow-up time was 92.0 months. Result At diagnosis, 91% of patients had at least one gene mutation with a median of 2.0 mutations (range 1-6) per patient; 49.4% had molecular gene mutations alone and 41.6% had both cytogenetic changes and molecular mutations. Mutations in NPM1, DNMT3A, NRAS and IDH2 were the most common mutations. According to the 2017 ELN recommendation, 49.3% of patients were in the favorable-risk group; 29.1%, the intermediate-risk group; and 21.6%, the unfavorable-risk group. Among the patients harboring at least one gene mutation at diagnosis, we randomly assigned them into the training (n=167) and validation cohort (n=111); the two cohorts had similar clinical features, and distribution of cytogenetic and molecular abnormalities. Based on the result from the analysis in the training cohort, we set 0.3% as the cut-off for MRD positivity because patients carried gene mutations lower than this limit had a similar outcome as those without detectable mutations. The allele frequencies of the mutants in MRD ranged from 0.3 to 50.5%. Excluding DTA mutations, 47.3% patients in the training cohort had MRD at 1st time point, and 26.9% at 2nd time point. The patients with positive NGS MRD had significantly higher relapse rate (P=0.042 for 1st MRD and P=0.035 for 2nd MRD), shorter disease-free survival (DFS, P=0.037 for 1st MRD and P=0.007 for 2nd MRD) and OS (P=0.015 for 1st MRD and P<0.001 for 2nd MRD, Figure 1). In multivariate Cox proportional hazards regression model incorporating age, white blood cell counts at diagnosis, transplantation status, 2017 ELN risk-stratification, number of chemotherapy cycles to attain CR, and the MRD status into analyses (Table 1), the 2nd MRD was an independent poor prognostic factor (P=0.040 for DFS and P=0.005 for OS) but not 1st MRD (P=0.113 for DFS and P=0.072 for OS). In the validation cohort, 2nd MRD positivity also predicted poorer OS and DFS (P=0.023 and P<0.001) but not 1st MRD (P=0.996 and P=0.461). A comparison of NGS with MCFC for the detection of MRD in 73 patients showed that MRD by NGS had significant additive prognostic value. Conclusion NGS-based MRD monitoring can be applied to more than 90% of AML patients who have detectable mutations at diagnosis. The presence of NGS MRD after treatment can predict outcome of AML patients, especially after the first consolidation chemotherapy (2nd MRD). Positivity of 2nd MRD is an independent unfavorable prognostic factor for DFS and OS. Further prospective trials are warranted to validate these findings and to clarify the role of pre-emptive treatment. Disclosures Tsai: Celgene: Research Funding; Astellas, BMS, Celgene, Chugai, Johnson & Johnson, Kirin, Novartis, Pfizer, Roche, Takeda: Honoraria. Tien:Novartis: Other: Travel Grant. Hou:Celgene: Research Funding; Abbvie, Astellas, BMS, Celgene, Chugai, Daiichi Sankyo, IQVIA, Johnson & Johnson, Kirin, Merck Sharp & Dohme, Novartis, Pfizer, PharmaEssential, Roche, Takeda: Honoraria. Tien:Celgene: Honoraria; Novartis: Honoraria; Alexion: Honoraria; BMS: Honoraria; Roche: Research Funding; Pfizer: Honoraria; Roche: Honoraria; Celgene: Research Funding; Abbvie: Honoraria; Johnson &Johnson: Honoraria; Daiichi Sankyo: Honoraria.
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Soverini, Simona, Caterina De Benedittis, Fausto Castagnetti, Gabriele Gugliotta, Manuela Mancini, Giorgina Specchia, Domenico Russo et al. « BCR-ABL Mutations in Chronic Myeloid Leukemia (CML) Patients (pts) with Failure and Warning to First- and Second-Line Tyrosine Kinase Inhibitor (TKI) Therapy : What Is the Advantage of Next-Generation Sequencing (NGS) over Conventional Sequencing ? » Blood 126, no 23 (3 décembre 2015) : 346. http://dx.doi.org/10.1182/blood.v126.23.346.346.
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Abstract Background - Point mutations in the BCR-ABL kinase domain are associated with resistance to TKI therapy. The most recent (2013) European Leukemia Net (ELN) recommendations have re(de)fined the criteria for failure in pts receiving 1st-line and 2nd-line TKI therapy and introduced the concept of warning. Assessing in how many CML patients with failure and warning mutations can be identified, especially now that more sensitive NGS-based mutation screening methods are available, would advance our knowledge of the biology of TKI resistance as well as contribute useful data to revise the ELN recommendations as to when and how BCR-ABL mutation analysis should be performed. Aims - We aimed to determine the frequency of BCR-ABL mutations as assessed by NGS vs conventional Sanger sequencing (SS) in CML pts with failure and warning to 1st- or 2nd-line TKI therapy as per the latest, 2013 ELN definitions. Methods - Between May 2013 and June 2015, 298 consecutive CML pts on TKI therapy were referred to our laboratory for BCR-ABL mutation screening by SS. One hundred and fifty-eight cases had no clinical data available, or were not in CP, or were receiving ≥3rd-line TKI therapy, or had confirmed/suspected nonadherence, or had experienced dose reductions for toxicity - leaving 140 pts who could be included in this study. Pts who were negative for mutations as determined by SS (n=105/140) were retrospectively reanalyzed by NGS on a Roche GS Junior, using a protocol already set up and optimized in the framework of the IRON II (Interlaboratory RObustness of NGS) international consortium. Sequencing depth allowed to achieve a lower mutation detection limit of 1% in all samples. Results - Failures and warnings to 1st-line therapy (imatinib, n=57; nilotinib, n=22; dasatinib, n=13) were 63 and 29, respectively. BCR-ABL mutations were found in 15/63 (24%) failures and 3/29 (10%) warnings by SS (Table 1). NGS reanalysis of the 74 pts with no evidence of mutations by SS revealed low burden (median, 6.6%; range, 1.5-11.7%) mutations in 6 failures and 1 warning, so that, overall, 21/63 (33%) failures and 4/29 (14%) warnings turned out to have mutations (Table 1). Mutations were E462K, E279K, K262R, F359I, E255K, F317L, K378R, A399T, L364I, V280A. No compound mutation was detected. Failures and warnings to 2nd-line therapy (nilotinib, n=27; dasatinib, n=21) were 35 and 13, respectively. SS identified mutations in 13/35 (37%) failures and 2/13 (15%) warnings (Table 1). NGS reanalysis of the 33 pts with no evidence of mutations by SS revealed low burden (median, 5.4%; range, 1.9-10.0%) mutations in 5 failures and 2 warnings, so that, overall, 18/35 (51%) failures and 4/13 (31%) warnings turned out to have mutations (Table 1). Mutations were T315I, E255V, F317I, E258D, P480L, Y393C, W261L, L370P, V371A, L324Q, again with no compound mutations. Table.All ptsPts positive for mutations by SSAdditional pts positive for mutations by NGSTotal pts positive for mutations1ST -LINE FAILURESNo CyR @ 3 mo9101BCR-ABL>10% @ 6 mo9000mCyR @ 6 mo1101BCR-ABL>1% @ 12 mo10022No CCyR @ 12 mo2101Loss of CCyR7314Loss of MMR20639Loss of CHR2101Progression to BP3202Total6315 (24%)621 (33%)1ST -LINE WARNINGSBCR-ABL>10% @ 3 mo7101BCR-ABL>1% @ 6 mo10112BCR-ABL>0.1% @ 12 mo12101Total293 (10%)14 (14%)2ND -LINE FAILURESNo CyR @ 3 mo3112BCR-ABL>10% @ 6 mo10224Loss of CCyR7303Loss of MMR6123Loss of CHR4303Progression to BP5303Total3513 (37%)518 (51%)2ND -LINE WARNINGSBCR-ABL>10% @ 3 mo6202BCR-ABL>0.1% @ 12 mo7022Total132 (15%)24 (31%) Conclusions 1) NGS allowed to identify BCR-ABL mutations in a greater proportion of cases as compared to SS. Low burden mutations included a T315I mutation in 2 pts on 2nd-line therapy classified as warnings: this would have turned them into failures. 2) Still, a substantial proportion of cases was found to not harbor any mutation, even when using a more sensitive NGS-based method. In particular, non-optimal achievement of the key molecular response milestones (10%, 1%, 0.1%) on 1st-line therapy was mostly not associated with BCR-ABL mutations, indicating that other mechanisms of molecular disease persistence have to be investigated in an attempt to optimize therapeutic outcomes. A national, multicenter study ('NEXT-IN-CML') aimed at the prospective assessment of NGS for routine BCR-ABL mutation screening of CML patients has just started. Supported by ELN, AIL, AIRC, FP7 NGS-PTL project, Progetto Regione-Università 2010-12 (L. Bolondi) Disclosures Soverini: Bristol-Myers Squibb: Consultancy; Ariad: Consultancy; Novartis: Consultancy. Castagnetti:BMS: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; ARIAD: Consultancy, Honoraria. Bonifacio:Ariad Pharmaceuticals: Consultancy; Amgen: Consultancy; Pfizer: Consultancy; Novartis Farma: Research Funding. Saglio:Bristol-Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; ARIAD: Consultancy, Honoraria; Novartis Pharmaceutical Corporation: Consultancy, Honoraria. Rosti:Novartis: Honoraria, Research Funding, Speakers Bureau; Bristol Myers Squibb: Honoraria, Research Funding, Speakers Bureau. Baccarani:NOVARTIS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; ARIAD Pharmaceuticals, Inc.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; PFIZER: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Martinelli:Pfizer: Consultancy; Novartis: Consultancy, Speakers Bureau; ROCHE: Consultancy; BMS: Consultancy, Speakers Bureau; AMGEN: Consultancy; MSD: Consultancy; Ariad: Consultancy.
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Kubaski, Francyne, Alberto Burlina, Giulia Polo, Danilo Pereira, Zackary M. Herbst, Camilo Silva, Franciele B. Trapp et al. « Experience of the NPC Brazil Network with a Comprehensive Program for the Screening and Diagnosis of Niemann-Pick Disease Type C ». International Journal of Neonatal Screening 8, no 3 (28 juin 2022) : 39. http://dx.doi.org/10.3390/ijns8030039.
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Niemann-Pick disease type C (NPC) is a lysosomal disorder caused by impaired cholesterol metabolism. Levels of lysosphingomyelin 509 (LysoSM509) have been shown elevated in dried blood spots (DBS) of NPC and acid sphingomyelinase deficiency patients. In this study, we report our experience using a two-tier approach (1st tier is the quantification of lysoSM509 by ultra-performance liquid chromatography tandem mass spectrometry followed by the 2nd tier with next-generation sequencing of the NPC1 and NPC2 genes). DBS samples from 450 suspected patients were received by the NPC Brazil network. Of these, 33 samples had elevated levels of lysoSM509, and in 25 of them, variants classified as pathogenic, likely pathogenic, or of unknown significance were identified in the NPC1 or NPC2 genes by next-generation sequencing. The quantification of lysoSM509 in DBS as a first-tier test for the diagnosis of NPC followed by molecular analysis of the NPC1 and NPC2 genes almost doubled the detection rate when compared to the performance of chitotriosidase activity as a first-tier biomarker, and it could likely be increased with the addition of a third tier with MLPA of the two genes involved. This strategy seems suitable for the neonatal screening (NBS) of NPC if this disease is eventually adopted by NBS programs.
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Soverini, Simona, Caterina De Benedittis, Luca Zazzeroni, Katerina Machova Polakova, Fausto Castagnetti, Gabriele Gugliotta, Maria Teresa Bochicchio et al. « In Chronic Myeloid Leukemia Patients on 2nd-Line Tyrosine Kinase Inhibitor Therapy, Deep Sequencing at the Time of Warning May Allow Sensitive Detection of Emerging BCR-ABL1 Mutants ». Blood 124, no 21 (6 décembre 2014) : 815. http://dx.doi.org/10.1182/blood.v124.21.815.815.
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Abstract Background and Aims: Next generation amplicon-based deep sequencing (DS) on the Roche, Illumina or Ion Torrent instruments is becoming accessible to a wider and wider number of diagnostic laboratories. Although conventional sequencing is still the gold standard, DS has been hailed by many as the future of diagnostic BCR-ABL1 kinase domain (KD) mutation screening. BCR-ABL1 KD mutations are infrequent in newly diagnosed chronic myeloid leukemia (CML) patients (pts) receiving 1st-line TKI therapy, but remain a challenge in relapsed pts, who usually display a greater genetic instability. Indeed, pts already harboring BCR-ABL1 KD mutations have a higher likelihood of developing additional, dasatinib (DAS)- or nilotinib (NIL)-resistant mutations – which is defined as a ‘failure’ by the 2013 European LeukemiaNet (ELN) recommendations. Taking advantage of a next-generation amplicon sequencing design and protocol set up and validated in the framework of the IRON-II international study, we aimed to assess whether DS may allow a larger window of detection of emerging BCR-ABL1 KD mutants predicting for an impending relapse. Methods: among the imatinib (IM)-resistant CML pts who switched to 2nd-line TKI therapy and were referred to our laboratory for routine BCR-ABL1 transcript level monitoring and KD mutation screening by conventional sequencing, 51 acquired DAS- or NIL-resistant mutations after a median of 9 months (range, 3-27 months) of therapy and had leftover cDNA available at previous timepoints. To reconstruct the dynamics of mutation emergence, resequencing on a Roche GS Junior instrument was performed from the time of failure and mutation detection by conventional sequencing backwards. Runs were designed to achieve high sequencing depth, allowing reliable detection of variants down to 1% abundance. BCR-ABL1/ABL1%IS transcript levels and/or cytogenetic response, whichever available, were used to define whether the patient had an ‘optimal response’, ‘warning’ or ‘failure’ at the time of first mutation detection by DS. Results: baseline mutation status, as assessed by conventional sequencing, was available for all the 51 CML pts included in this retrospective study; 29/51 pts were positive for BCR-ABL1 KD mutations, with switch to NIL or DAS selected accordingly. Twenty-six pts were later found to have acquired DAS-resistant mutations (T315I, n=13; F317L/V, n=10; V299L, n=3) and 25 pts were later found to have acquired NIL-resistant mutations (T315I, n=4; F359V/I/C, n=7; Y253H, n=6; E255K, n=9; one patient acquired two mutations). DS was able to backtrack the DAS- or NIL-resistant mutations to the previous sample(s) in 23/51 (45%) pts. Median mutation burden at the time of first detection by DS was 5% (range, 1-17%); median interval between detection by DS and detection by conventional sequencing was 3 months (range, 3-9 months). In 5 cases, the mutations were traceable at baseline; in the remaining cases, correlation with response at the time mutations were first detected by DS revealed a ‘warning’ according to the 2013 ELN definitions of response to 2nd-line therapy in 13 cases; an ‘optimal response’ in one case; a ‘failure’ in 4 cases. As a control, we used DS to explore BCR-ABL1 KD mutation status in 10 randomly selected pts with ‘warning’ at various timepoints, that later turned into optimal responses; no DAS- or NIL-resistant mutations were detected. Conclusions: the 2011 ELN recommendations for mutation analysis suggest BCR-ABL1 KD to be screened by conventional sequencing in case of ‘failure’ of 2nd-line TKI therapy – according to the provisional definitions available at the time. Earlier detection of emerging BCR-ABL1 KD mutations allows a greater leeway in tackling drug resistance and enhancing therapeutic efficacy. Data presented herein indicate that: 1) DS may reliably pick TKI-resistant mutations earlier than conventional sequencing in a proportion of pts, and that 2) the recently introduced definitions of ‘warning’ may provide a rational trigger, besides ‘failure’, for DS-based BCR-ABL1 KD mutation screening in CML pts on 2nd-line TKI therapy. A prospective cost-benefits evaluation of using DS in this and other settings is warranted, and will contribute useful information to the revision of the ELN recommendations for BCR-ABL1 KD mutation analysis. Supported by: European LeukemiaNet, AIL, AIRC, FP7 NGS-PTL project, Progetto Regione-Università 2010-12 (L. Bolondi). Disclosures Soverini: Novartis: Consultancy; Bristol-Meyers Squibb: Consultancy; Ariad: Consultancy. Castagnetti:Novartis Farma: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy. Gugliotta:Novartis: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria. Bonifacio:Amgen Inc.: Consultancy. Rosti:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy. Baccarani:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; Ariad: Consultancy; Pfizer: Consultancy. Martinelli:NOVARTIS: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.
Thèses sur le sujet "2nd generation sequencing"
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Dal, Molin Alessandra. « Structural annotation of eukaryotic genomes in 2nd generation sequencing era ». Doctoral thesis, 2016. http://hdl.handle.net/11562/940817.
Texte intégralRésumé :
Nell'ultimo decennio, l'aumento dell’efficienza e la diminuzione del costo delle nuove tecniche di sequenziamento ha portato ad un accumulo di sequenze genomiche nei database pubblici. Con questa enorme quantità di sequenze a disposizione, la necessità di generare delle annotazioni precise e dettagliate non è mai stata così grande. L’annotazione strutturale del genoma è il processo di identificazione di elementi strutturali in una sequenza di DNA, classificandoli in base al loro ruolo biologico. L’approccio computazionale viene sempre più utilizzato per poter eseguire l'annotazione strutturale in maniera automatica, con tempi di esecuzione generalmente brevi e che soddisfano le esigenze ‘high-throughput’ dei progetti di sequenziamento del genoma, anche se sono meno precisi rispetto alla cura manuale, che rimane il 'golden standard' per valutare l’affidabilità dell’annotazione prodotta.Lo scopo di questo progetto è quello di produrre un’annotazione del genoma in maniera veloce ed accurata, in linea con le esigenze attuali, mediante un approccio computazionale applicato a diversi casi sperimentali, in base alle conoscenze biologiche di base e alla natura dei dati di partenza. Per la completezza dello studio, il contributo dei vari metodi utilizzati per produrre l'annotazione finale è stato analizzato insieme alla valutazione della qualità dei dati prodotti.I risultati ottenuti hanno confermato il fatto che la complessità dei genomi eucariotici influisce notevolmente sul processo di annotazione. Una vasta porzione di geni può essere annotata grazie principalmente all’omologia con geni o proteine di altre specie evolutivamente vicine, oppure con l'utilizzo di predittori ab initio ed evidenze sperimentali specie-specifiche. L'integrazione di molteplici evidenze migliora notevolmente l'accuratezza delle annotazioni finali, tuttavia la valutazione della qualità dei risultati e il filtraggio di sequenze poco affidabili, insieme alla cura manuale, sono tuttora necessari per garantire un risultato ottimale.In the last decade the increase in efficiency and decrease in cost of new sequencing techniques led to a growing amount of genomic sequences in publicdatabases. With this huge volume of sequences being generated from highthroughput sequencing projects, the requirement for providing accurate anddetailed genome annotations has never been greater. Structural genome annotation is the process of identifying structural features in a DNA sequence and classifying them based on their biological role. Computer programs are increasingly used to perform structural annotation since they meet the high-throughput demands of genome sequencing projects even if they are less accurate than manual gene annotation which remains the ‘golden-standard’ for evaluating annotation confidence and quality.The aim of this project is to meet the need of producing fast and accurate genome annotation by applying available computational means to different experimental cases, depending on the biological knowledge achieved so far and the quality of starting data. The contribution of different methods used to produce the final annotation has been analyzed along with the evaluation of results for the completeness of the study.The results obtained showed that the complexity of eukaryotic genomes greatly affects the annotation process; a big fraction of the genes in a genome sequence can be found mostly by homology to other known genes or proteins and by the use of ab initio predictors and species-specific evidence. The integration of multiple sources of annotation greatly improved the accuracy of the final genome annotations, anyway being not error free. Quality assessment of results and filtering of low confidence sequences together with manual revision are Always required to achieve higher accuracy.
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Minio, Andrea. « Genome Assembly With 2nd Generation Sequencing Technologies : Definition of Best Experimental Design In Relation To Genomic Features ». Doctoral thesis, 2015. http://hdl.handle.net/11562/915782.
Texte intégralRésumé :
L'avvento della Seconda Generazione delle tecnologie di sequenziamento ha cambiato profondamente il processo di generazione di dati a partire dalle molecole di DNA, che è diventato più economico e più veloce. La varietà di tecnologie e strumenti per la ricostruzione disponibili, ognuna con differenti punti di forza e debolezze, rende difficoltoso il compito di definire il set-up sperimentale ottimale per ricostruire il genoma di una nuova specie.
In questo lavoro è stata affrontata la ricostruzione di genomi di diverse specie con molteplici strategie. Questo ha reso possibile definire dei protocolli che ottimizzano costi e risultati in funzione delle caratteristiche genetiche dell’oggetto dello studio.
Nel caso di organismi batterici, la ridotta lunghezza e la bassa complessità della sequenza genetica permette di ottenere risultati di alta qualità anche utilizzando una singola libreria Illumina standard, in maniera indipendente da quale algoritmo si decida di utilizzare per la ricostruzione.
Genomi fungini mostrano invece, assieme ad una lunghezza maggiore, un’aumentata complessità rispetto ai genomi di procarioti. L’uso di librerie standard Illumina da sole non consente di eliminare il problema della frammentazione e l’ottimizzazione delle procedure bioinformatiche migliora solo marginalmente i risultati. L’aggiunta di librerie Mate Pair oppure il sequenziamento con tecnologia PacBio sono due alternative che portano, con costi similari, a risultati di alta qualità.
I genomi delle piante oltre ad essere essere più lunghi presentano li maggiore grado di complessità, con un elevato contenuto in ripetizioni e alti tassi di eterozigosità. Le librerie standard di Illumina, a causa della ridotta lunghezza dei frammenti sequenziati, non possono ridurre il problema della frammentazione delle sequenze consenso. Le librerie Mate Pair, invece, riescono a ridurre il problema, le più lunghe superano ripetizioni più estese mentre le più corte migliorano al ricostruzione del contenuto dei gap. La tecnologia PacBio ha dimostrato di essere una soluzione efficace nella riduzione della frammentazione, ma il costo proibitivo ne impedisce l’uso da sola. L’assemblaggio ibrido è una possibile alternativa combinando un’elevata profondità di sequenziamento di reads Illumina, corte ma economiche e affidabili, con una limitata quantità di reads PacBio, lunghe ma con un alto tasso d’errore. Questa soluzione porta ad una riduzione dei costi di sequenziamento ma anche a risultati di qualità inferiore, in aggiunta le risorse computazionali necessarie crescono a dismisura.
Sono disponibili molteplici soluzioni al problema della ricostruzione della sequenza di un genoma ma sono le caratteristiche del genoma stesso che indicano la combinazione di tecnologia di sequenziamento e procedimento informatico che meglio ottimizzano i costi e le qualità dei risultati ottenibili.The advent of the Second Generation of sequencing technologies deeply changed the process of generating data from DNA molecules, which has become cheaper and faster. The multiplicity of technologies and assembly tools available, each with different strengths and weaknesses, turns the choice of a proper experimental set-up when approaching the genome of a new species into a difficult task. In this work, multiple strategies have been adopted for reconstructing the genomes of different species. This has allowed profiling of the practices that best optimize costs and results according to the genetic characteristics of the subject of study. When dealing with bacterial organisms, the short genome length and a low complexity of the underlying sequence allows to obtain a high quality draft even when using only one standard Illumina library - regardless of the assembly procedure adopted. Fungal genomes show an increased length and a higher complexity when compared with prokaryotic organisms. Standard Illumina libraries are not sufficient to overcome the fragmentation issue of the draft sequence, and improving the computational assembly pipeline shows only a limited power in ameliorating the results. Additional Mate Pair sequencing data or PacBio long reads sequencing can be adequate alternatives, as they both lead to high quality assembly results at similar expenses. Long plant genomes show the highest complexity degree, with an elevated repetitive content and high heterozygosity rate. Standard Illumina libraries are not sufficient to overcome the fragmentation problem due to the limited insert size. Mate pair sequences greatly improve the results, with longer libraries spanning longer repeats and shorter ones improving the gap reconstruction. PacBio showed to be an effective solution to this problem, but given its high sequencing costs it is prohibitive to adopt this technology alone for reconstruction. Hybrid assembly is a possible alternative, combining an high coverage of Illumina short but cheap and reliable reads with a low coverage of longer but more erroneous PacBio reads. This solution has lower sequencing costs, but the quality of the results is limited by the coverage of long reads; moreover, the computational resources necessary to perform error correction and assembly are massively increased. -4- When approaching the reconstruction of a genome, therefore, multiple solutions are available – but it is the available knowledge of its characteristics that indicates the best combination of assembly tools and sequencing technologies to optimise both expenses and quality of the results.
Chapitres de livres sur le sujet "2nd generation sequencing"
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Andrews, Jonathan C., Michael F. Wangler, Shinya Yamamoto et Jennifer E. Posey. « Advances in Next-Generation Sequencing Technologies and Functional Investigation of Candidate Variants in Neurological and Behavioral Disorders ». Dans Encyclopedia of Behavioral Neuroscience, 2nd edition, 390–404. Elsevier, 2022. http://dx.doi.org/10.1016/b978-0-12-819641-0.00145-6.
Texte intégralActes de conférences sur le sujet "2nd generation sequencing"
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Liu, Xuan, Shing-Wan Choi, Thomas K. F. Wong, Tak-Wah Lam et Siu-Ming Yiu. « Detection of novel tandem duplication with next-generation sequencing ». Dans the 2nd ACM Conference. New York, New York, USA : ACM Press, 2011. http://dx.doi.org/10.1145/2147805.2147861.
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Gao, Jingyang, Fei Qi et Rui Guan. « Structural variation discovery with next-generation sequencing ». Dans 2013 2nd International Symposium on Instrumentation & Measurement, Sensor Network and Automation (IMSNA). IEEE, 2013. http://dx.doi.org/10.1109/imsna.2013.6743374.
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Johnson, Daniel, Kun Wang, Carole L. Cramer et Xiuzhen Huang. « Graph-based approach for gene markers and applications in next-generation sequencing data analysis ». Dans the 2nd ACM Conference. New York, New York, USA : ACM Press, 2011. http://dx.doi.org/10.1145/2147805.2147886.
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Gribchenko, E. S. « The study of transcriptomes of symbiotic tissue of pea using the third-generation sequencing technology Oxford Nanopore ». Dans 2nd International Scientific Conference "Plants and Microbes : the Future of Biotechnology". PLAMIC2020 Organizing committee, 2020. http://dx.doi.org/10.28983/plamic2020.093.
Texte intégralRésumé :
The transcriptome profiles the cv. Frisson mycorrhizal roots and inoculated nitrogen-fixing nodules were investigated using the Oxford Nanopore sequencing technology. A database of gene isoforms and their expression has been created.
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Sakarya, Onur, et Heinz Breu. « Workshop : Gene fusion detection with short and medium length next generation sequencing ». Dans 2012 IEEE 2nd International Conference on Computational Advances in Bio and Medical Sciences (ICCABS). IEEE, 2012. http://dx.doi.org/10.1109/iccabs.2012.6182676.
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Ma, Xuezheng, Shuru Chen, Liping Zhang, Hong Deng, Lili Li, Fangji Yang, Pengfei Yang, Pengchen Yan et Kongxin Hu. « Next generation sequencing and bioinformatic approaches to identify dengue viruses from non-invasive clinical samples ». Dans 2017 2nd IEEE International Conference on Computational Intelligence and Applications (ICCIA). IEEE, 2017. http://dx.doi.org/10.1109/ciapp.2017.8167246.
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O'Neill, Rachel, Ion Mandoiu, Mazhar I. Khan, Craig Obergfell, Hongjun Wang, Andrew Bligh, Bassam Tork, Nicholas Mancuso et Alexander Zelikovsky. « Workshop : Bioinformatics methods for reconstruction of Infectious Bronchitis Virus quasispecies from next generation sequencing data ». Dans 2012 IEEE 2nd International Conference on Computational Advances in Bio and Medical Sciences (ICCABS). IEEE, 2012. http://dx.doi.org/10.1109/iccabs.2012.6182674.
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« Sequencing and iterative assembly of Ixiolirion tataricum plastome from total DNA using 2nd and 3rd generation HTS platforms ». Dans Plant Genetics, Genomics, Bioinformatics, and Biotechnology. Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, 2019. http://dx.doi.org/10.18699/plantgen2019-131.
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Sadikin, Rifki, Andria Arisal, Rofithah Omar et Nur Hidayah Mazni. « Processing next generation sequencing data in map-reduce framework using hadoop-BAM in a computer cluster ». Dans 2017 2nd International Conferences on Information Technology, Information Systems and Electrical Engineering (ICITISEE). IEEE, 2017. http://dx.doi.org/10.1109/icitisee.2017.8285542.
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Lalinska-Volekova, Bronislava, Hana Majerova, Ivona Kautmanova, Tomas Farago, Dana Szaboova et Jana Brcekova. « MICROBIAL COMPOSITION OF NATURAL Fe OXYHYDROXIDES AND ITS INFLUENCE ON ARSENIC AND ANTIMONY SORPTION ». Dans 22nd SGEM International Multidisciplinary Scientific GeoConference 2022. STEF92 Technology, 2022. http://dx.doi.org/10.5593/sgem2022/5.1/s20.037.
Texte intégralRésumé :
The presented paper represents a comprehensive analysis of hydrous ferric oxides (HFOs) precipitated from Fe rich drainage waters contaminated by arsenic and antimony. Ochre samples from three abandoned Sb deposits were collected in three different seasons and were characterized from the mineralogical, geochemical, and microbiological point of view. They were formed mainly by poorly crystallized 2-line ferrihydrite, with the content of arsenic in samples ranging from 0.7 wt.% to 13 wt.% and content of antimony ranging from 0.025 wt.% up to 1.2 wt.%. Next-generation sequencing approach with 16S RNA, 18S RNA and ITS markers was used to characterize bacterial, fungal, algal, metazoal and protozoal communities occurring in the HFOs. In the 16S RNA, the analysis dominated bacteria (96.2 %) were mainly Proteobacteria (68.8 %) and Bacteroidetes (10.2 %) and to less extent also Acidobacteria, Actinobacteria, Cyanobacteria, Firmicutes, Nitrosprae and Chloroflexi. Alpha and beta diversity analysis revealed that the bacterial communities of individual sites do not differ significantly, and only subtle seasonal changes were observed. This study provides evidence of diverse microbial communities that exist in drainage waters and are highly important in the process of mobilization or immobilization of the potentially toxic elements.