scholarly journals Massively parallel sequencing and genome-wide copy number analysis revealed a clonal relationship in benign metastasizing leiomyoma

Oncotarget ◽  
2017 ◽  
Vol 8 (29) ◽  
pp. 47547-47554 ◽  
Author(s):  
Ren-Chin Wu ◽  
An-Shine Chao ◽  
Li-Yu Lee ◽  
Gigin Lin ◽  
Shu-Jen Chen ◽  
...  
Keyword(s):  
Copy Number ◽  
Massively Parallel Sequencing ◽  
Massively Parallel ◽  
Copy Number Analysis ◽  
Benign Metastasizing Leiomyoma ◽  
Parallel Sequencing ◽  
Clonal Relationship ◽  
Genome Wide ◽  
Metastasizing Leiomyoma

scholarly journals Genome-Wide Massively Parallel Sequencing of Formaldehyde Fixed-Paraffin Embedded (FFPE) Tumor Tissues for Copy-Number- and Mutation-Analysis

PLoS ONE ◽  
2009 ◽  
Vol 4 (5) ◽  
pp. e5548 ◽  
Author(s):  
Michal R. Schweiger ◽  
Martin Kerick ◽  
Bernd Timmermann ◽  
Marcus W. Albrecht ◽  
Tatjana Borodina ◽  
...  
Keyword(s):  
Mutation Analysis ◽  
Copy Number ◽  
Massively Parallel Sequencing ◽  
Massively Parallel ◽  
Parallel Sequencing ◽  
Genome Wide ◽  
Tumor Tissues ◽  
Ffpe Tumor

Whole Genome Paired End Sequencing Identifies Genomic Evolution in Myeloma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2846-2846 ◽  
Author(s):  
Nikhil C. Munshi ◽  
Herve Avet-Loiseau ◽  
Phil Stephens ◽  
Masood A Shammas ◽  
Cheng Li ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Genetic Instability ◽  
Copy Number ◽  
Research Funding ◽  
Massively Parallel Sequencing ◽  
Massively Parallel ◽  
Advisory Committees ◽  
Parallel Sequencing ◽  
Genome Wide ◽  
Paired End Sequencing

Abstract Abstract 2846 Poster Board II-822 Background: A prominent feature of most cancers is striking genetic instability and ongoing accrual of mutational changes associated with tumor progression, including acquisition of invasiveness, drug resistance, and metastasis. Methods: We first utilized single nucleotide polymorphism (SNP) arrays (Affymetrix) to evaluate genome-wide gains and losses in copy number and heterozygosity in CD138+ multiple myeloma (MM) cells collected from 14 patients at two time points at least 6 months apart. To estimate the extent of genomic instability in each patient, the number of events leading to copy number or heterozygosity changes throughout the genome were calculated. An event was defined as detectable change in copy number or heterozygosity in three or more consecutive SNPs. Two cases were also investigated for genome-wide rearrangements utilizing a paired-end approach on next generation sequencing. Results: In a period of six months, all MM patients analyzed acquired multiple new mutational events including changes in copy number and heterozygosity, ranging from 0.021 - 2.674 %, indicating a wide range of genetic instability. Although the rate of mutation varied, the majority (71%) of MM patients had acquired > 100 mutational events within the six months period, thus indicating a striking genetic instability. Chromosomes 1, 13, and X were unique with respect to copy number changes and showed large areas of change, spanning the entire length of a chromosome in several patient samples analyzed. Chromosomes 1 and 13 also showed large areas of loss or gain of heterozygosity in several patients, indicating areas of recurrent changes. We were also able to correlate genomic changes with changes in expression of corresponding genes. In two cases, we investigated genome-wide rearrangements utilizing a massively parallel sequencing approach. Short insert (400bp) libraries from two samples collected 6 months apart were constructed and subjected to paired-end sequencing utilizing 37bp readlengths on the Illumina GAII instrument. Approximately 80 million reads were generated for each of the 4 samples. Read pairs were mapped back to the reference genome, and those mapping aberrantly (incorrect orientation, different chromosomes, incorrect genomic distance) were further analyzed. Bespoke PCR assays defining each breakpoint were designed and used to verify the somatic nature of the mapped rearrangement. Further, PCR fragments spanning somatic genomic rearrangements were sequenced to generate base-pair resolution of breakpoints. To date, 29 somatic rearrangements have been sequenced, including three that were present only in the second sample. One of these was on chromosome 13. Breakpoint sequencing revealed a 64.9Kb homozygous (no wild-type readpairs found) deletion removing the first two exons of the RB1 gene. No reads spanning this breakpoint were found in the matching sample taken six months earlier. Conclusions: This is the first study utilizing massively parallel sequencing to investigate the MM genome and provides important insight into the pathogenesis of disease progression .as well as confirms the potential of whole genome sequencing to inform biology of the disease that may affect the therapeutic approach in future. Disclosures: Munshi: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis : Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Richardson:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millennium Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Anderson:Celgene: Consultancy, Honoraria, Research Funding; Millennium: Consultancy, Honoraria, Research Funding; Novartis : Consultancy, Honoraria, Research Funding.

Comprehensive preimplantation genetic testing by massively parallel sequencing

2020 ◽  
Author(s):  
Songchang Chen ◽  
Xuyang Yin ◽  
Sijia Zhang ◽  
Jun Xia ◽  
Ping Liu ◽  
...  
Keyword(s):  
Genetic Testing ◽  
Genome Sequencing ◽  
Massively Parallel Sequencing ◽  
Science And Technology ◽  
Family Members ◽  
Snp Array ◽  
Massively Parallel ◽  
Preimplantation Genetic Testing ◽  
Parallel Sequencing ◽  
Genome Wide

Abstract STUDY QUESTION Can whole genome sequencing (WGS) offer a relatively cost-effective approach for embryonic genome-wide haplotyping and preimplantation genetic testing (PGT) for monogenic disorders (PGT-M), aneuploidy (PGT-A) and structural rearrangements (PGT-SR)? SUMMARY ANSWER Reliable genome-wide haplotyping, PGT-M, PGT-A and PGT-SR could be performed by WGS with 10× depth of parental and 4× depth of embryonic sequencing data. WHAT IS KNOWN ALREADY Reduced representation genome sequencing with a genome-wide next-generation sequencing haplarithmisis-based solution has been verified as a generic approach for automated haplotyping and comprehensive PGT. Several low-depth massively parallel sequencing (MPS)-based methods for haplotyping and comprehensive PGT have been developed. However, an additional family member, such as a sibling, or a proband, is required for PGT-M haplotyping using low-depth MPS methods. STUDY DESIGN, SIZE, DURATION In this study, 10 families that had undergone traditional IVF-PGT and 53 embryos, including 13 embryos from two PGT-SR families and 40 embryos from eight PGT-M families, were included to evaluate a WGS-based method. There were 24 blastomeres and 29 blastocysts in total. All embryos were used for PGT-A. Karyomapping validated the WGS results. Clinical outcomes of the 10 families were evaluated. PARTICIPANTS/MATERIALS, SETTING, METHODS A blastomere or a few trophectoderm cells from the blastocyst were biopsied, and multiple displacement amplification (MDA) was performed. MDA DNA and bulk DNA of family members were used for library construction. Libraries were sequenced, and data analysis, including haplotype inheritance deduction for PGT-M and PGT-SR and read-count analysis for PGT-A, was performed using an in-house pipeline. Haplotyping with a proband and parent-only haplotyping without additional family members were performed to assess the WGS methodology. Concordance analysis between the WGS results and traditional PGT methods was performed. MAIN RESULTS AND THE ROLE OF CHANCE For the 40 PGT-M and 53 PGT-A embryos, 100% concordance between the WGS and single-nucleotide polymorphism (SNP)-array results was observed, regardless of whether additional family members or a proband was included for PGT-M haplotyping. For the 13 embryos from the two PGT-SR families, the embryonic balanced translocation was detected and 100% concordance between WGS and MicroSeq with PCR-seq was demonstrated. LIMITATIONS, REASONS FOR CAUTION The number of samples in this study was limited. In some cases, the reference embryo for PGT-M or PGT-SR parent-only haplotyping was not available owing to failed direct genotyping. WIDER IMPLICATIONS OF THE FINDINGS WGS-based PGT-A, PGT-M and PGT-SR offered a comprehensive PGT approach for haplotyping without the requirement for additional family members. It provided an improved complementary method to PGT methodologies, such as low-depth MPS- and SNP array-based methods. STUDY FUNDING/COMPETING INTEREST(S) This research was supported by the research grant from the National Key R&D Program of China (2018YFC0910201 and 2018YFC1004900), the Guangdong province science and technology project of China (2019B020226001), the Shenzhen Birth Defect Screening Project Lab (JZF No. [2016] 750) and the Shenzhen Municipal Government of China (JCYJ20170412152854656). This work was also supported by the National Natural Science Foundation of China (81771638, 81901495 and 81971344), the National Key R&D Program of China (2018YFC1004901 and 2016YFC0905103), the Shanghai Sailing Program (18YF1424800), the Shanghai Municipal Commission of Science and Technology Program (15411964000) and the Shanghai ‘Rising Stars of Medical Talent’ Youth Development Program Clinical Laboratory Practitioners Program (201972). The authors declare no competing interests. TRIAL REGISTRATION NUMBER N/A.

Global Identification of Genomic Structural Variants In Childhood ETV6/RUNX1 (TEL/AML1) Acute Lymphoblastic Leukemias by Mate-Pair Massively Parallel Sequencing.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3364-3364
Author(s):  
Vera Okpanyi ◽  
Christoph Bartenhagen ◽  
Michael Gombert ◽  
Vera Binder ◽  
Hans-Ulrich Klein ◽  
...  
Keyword(s):  
Fusion Gene ◽  
Massively Parallel Sequencing ◽  
Chromosomal Rearrangements ◽  
Massively Parallel ◽  
Structural Variants ◽  
Structural Variations ◽  
Parallel Sequencing ◽  
Clonal Relationship ◽  
Mate Pair ◽  
Leukemic Clone

Abstract Abstract 3364 Introduction Chimeric fusion genes generated by chromosomal translocations are highly prevalent in childhood acute lymphoblastic leukemia (ALL) and mainly represent early, prenatal events. The t(12;21)(p13;q22) translocation generates the ETV6/RUNX1 fusion gene and is the most frequent gene recombination in childhood B-lineage ALL, occurring in approximately 25% of the cases. It is associated with favorable prognosis even though a substantial proportion of the cases relapse. The fusion gene itself initiates the pre-leukemic process but additional genetic hits are needed to trigger a full blown leukemia. Being one of the best-characterized childhood leukemias, both nature and mechanisms of the events that cooperate with the chimeric protein are still poorly understood. Furthermore, studies addressing the genetic origin of relapse demonstrated a clonal relationship between relapse and diagnostic sample, assuming the existence of an ancestral, pre-leukemic clone. Objective Second-generation sequencing of both ends of huge numbers of DNA fragments allows comprehensive characterization of patterns of somatic rearrangements on an unprecedented, high-resolution level. By using the Illumina mate-pair massively parallel sequencing technology and intra-individual side-by-side comparison of leukemic and normal germline DNA, we aim to elucidate the cooperating genetic events in leukemogenesis in ETV6/RUNX1-ALLs as well as the clonal relationship between relapse and diagnostic sample. Methods We investigated diagnostic and relapse samples as well as non-leukemic germline material from one pediatric patient, diagnosed with ETV6/RUNX1-ALL in Germany. Mate-pair genomic sequencing libraries with an insert size of approximately 2-kb were constructed and paired-end sequence reads of 36-bp each were generated on the Illumina Genome Analyzer IIx from randomly created ~500-bp DNA fragments. Data were filtered and aligned to the human reference genome (GRCh37) using BWA. Reads considered PCR duplicates were removed and detection as well as clustering of structural variants (translocations, deletions, inversions) was subsequently carried out with GASV. Discordantly mapping read pairs defined potential structural variations and cluster sizes of at least 4 uniquely and correctly mapping read pairs were included in further analyses. In order to confirm breakpoints and resolve them to base-pair level, areas of putative chromosomal rearrangements were amplified from genomic DNA of tumor and matched normal sample and were conventionally sequenced. Results An average of ~73,000,000 read pairs were generated for each sample and after alignment, the whole genome was sequenced with a mean fragment coverage of 18.8X. A substantial variation in prevalence of structural variants could be detected between paired diagnostic and relapse samples. Within the diagnostic sample we could in total observe 739 deletions, 66 inversions and 107 translocations while the relapse sample exhibited 26 deletions, 14 inversions and 240 translocations. In a first analysis we focused on translocations, presuming the high impact of chromosomal rearrangements on leukemogenesis. Subtracting translocations being of germline origin, 73 translocations at diagnosis and 207 translocations in relapse could be detected, of which 183 (74%) were identified being intragenic. Remarkably, both samples shared only 16 translocations (6%), while 57 (22%) uniquely appear in the diagnostic sample and 191 (72%) could only be observed in the relapse sample. Intragenic, shared translocations in diagnostic and relapse samples include the t(12;24) PDE3A/RN18S1, the t(2;17) PID1/UNC45B as well as the t(1;6) HFM1/EYA4 fusion gene products. Detection of the known ETV6/RUNX1 translocation in diagnostic and relapse sample as well as confirmation of selected breakpoints via Sanger sequencing validated our methodological approach. Conclusion Mate-pair sequencing of leukemic samples in comparison to germline material provides a powerful tool to identify genome-wide chromosomal structural variations and will allow analysis of clonality between diagnostic and relapse samples. The low percentage of shared translocations gives a first hint probably objecting the thesis of a common pre-leukemic clone, but will only be elucidated by analysis of further patient samples. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Cancer Genome Scanning in Plasma: Detection of Tumor-Associated Copy Number Aberrations, Single-Nucleotide Variants, and Tumoral Heterogeneity by Massively Parallel Sequencing

2013 ◽  
Vol 59 (1) ◽  
pp. 211-224 ◽  
Author(s):  
KC Allen Chan ◽  
Peiyong Jiang ◽  
Yama WL Zheng ◽  
Gary JW Liao ◽  
Hao Sun ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
Cancer Patients ◽  
Copy Number ◽  
Massively Parallel Sequencing ◽  
Point Mutations ◽  
Cancer Genome ◽  
Massively Parallel ◽  
Plasma Dna ◽  
Parallel Sequencing ◽  
Copy Number Aberrations

BACKGROUND Tumor-derived DNA can be found in the plasma of cancer patients. In this study, we explored the use of shotgun massively parallel sequencing (MPS) of plasma DNA from cancer patients to scan a cancer genome noninvasively. METHODS Four hepatocellular carcinoma patients and a patient with synchronous breast and ovarian cancers were recruited. DNA was extracted from the tumor tissues, and the preoperative and postoperative plasma samples of these patients were analyzed with shotgun MPS. RESULTS We achieved the genomewide profiling of copy number aberrations and point mutations in the plasma of the cancer patients. By detecting and quantifying the genomewide aggregated allelic loss and point mutations, we determined the fractional concentrations of tumor-derived DNA in plasma and correlated these values with tumor size and surgical treatment. We also demonstrated the potential utility of this approach for the analysis of complex oncologic scenarios by studying the patient with 2 synchronous cancers. Through the use of multiregional sequencing of tumoral tissues and shotgun sequencing of plasma DNA, we have shown that plasma DNA sequencing is a valuable approach for studying tumoral heterogeneity. CONCLUSIONS Shotgun DNA sequencing of plasma is a potentially powerful tool for cancer detection, monitoring, and research.

Sign in / Sign up

Export Citation Format

Share Document