scholarly journals Family Trio-Based Whole Genome Optical Mapping Identifies Candidate Structural Variations Predisposing Children to Acute Lymphoblastic Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5201-5201
Author(s):  
Ute Fischer ◽  
Layal Yasin ◽  
Julia Täubner ◽  
Triantafyllia Brozou ◽  
Arndt Borkhardt
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Family History ◽  
De Novo ◽  
Lymphoblastic Leukemia ◽  
Germline Mutations ◽  
Whole Genome ◽  
Structural Variations ◽  
Short Read ◽  
Short Read Sequencing ◽  
Family Trio

Germline mutations account for a substantial proportion of childhood cancer and may critically affect disease characteristics, therapy efficacy, severity of treatment side effects and patient outcome. To date, only 8-10% of childhood cancer cases can be explained by germline mutations identified in known cancer predisposing genes. This is in part due to the technical limitation of next generation short read sequencing, which detects single nucleotide variants, small deletions/insertions or simple copy number variations, but is not a reliable tool to identify larger structural variations (SVs, >500 bp) which are frequent in the human genome and may impact on disease predisposition. Using whole genome optical mapping (WGOM) we aimed at identification of de novo and inherited germline SVs in a cohort of patients with clinically suspected cancer predisposition but without informative findings in short read sequencing analyses. After informed consent we performed family trio based short read (2x 100 bp) whole exome sequencing (WES) on a HiSeq2500 (Illumina) and collected clinical and demographic data for a cohort of >100 families with children affected by cancer who were treated in our hospital. About 25% of the patients either (1) had a family history indicative of cancer susceptibility, or (2) had accompanying clinical findings (e.g. developmental delay, congenital anomalies) or (3) experienced excessive toxicity during chemotherapy. From this subgroup we selected four patients with acute lymphoblastic leukemia whose sequencing data and routine genetic workup were not informative of a known cancer predisposing syndrome and employed family trio-based next generation WGOM on a Saphyr instrument equipped with Access software (Bionano Genomics) to identify genomic SVs. To this end, we extracted and labeled high molecular weight DNA molecules at specific hexamer sequence motifs (average distance: 5 kb) using a DNA methyltransferase-based direct labeling reaction. Imaging was carried out on single-molecule level and each sample genome was de novo assembled from molecule data. Consensus genome maps were clustered into two alleles and diploid assemblies created. Genomes of patients were compared to parental genomes and the GRCh38 reference genome. SVs were inferred from de novo assemblies and genome comparisons with respect to quality scores, overall molecule coverage, fraction of molecules displaying the SV event, and chimeric DNA fragment mapping. Specific SV calls were compared to a set of > 160 human control samples (provided by Bionano Genomics) to filter against common SVs and potential artifacts. Filtered SVs were annotated using structural variant and gene databases. Employing WGOM we analyzed DNA molecules 300.000 bp long on average and achieved genomic coverage ranging from 90-132x corresponding to 330-480 Gbp. For instance, for one patient, we obtained 1751 insertions, 624 deletions, 77 inversions, 21 duplications, 1 intra- and 2 inter-chromosomal translocations before filtering. The majority of these events (78%) were inherited from both parents. 20% were inherited from either father or mother and 2% were generated de novo. As the family history of this patient was inconspicuous for tumor diseases, we removed all inherited events and filtered against common variants. This resulted in only two candidate de novo lesions: a heterozygous 129,495 bp deletion framed by inversions (chr9: 66,156,733-66,622,623) in a gene-less region and a heterozygous inverted 352,667 bp duplication (chr22: 15,522,454-15.875,120) that spanned the genes OR11H, POTEH, POTEH-AS1, LINC01297, DUXAP8, and BMS1P22. Of these genes DUXAP8 is an oncogenic non-coding RNA of the homeobox gene family that has been associated with increased tumor growth and poorer prognosis in a wide variety of somatic cancers. It functions as a regulator of transcription by binding to key components of the developmental regulator epigenetic polycomb repressive complex 2 and may thus account for additional presentations of the child (dwarfism, accelerated skeletal age, linguistic developmental delay, morphological traits). Our results indicate that WGOM is a useful technology to identify candidate SVs in children predisposed to cancer and developmental syndromes. Several candidates are currently being tested and the results will be presented. Disclosures No relevant conflicts of interest to declare.

scholarly journals Resolving the Full Spectrum of Human Genome Variation using Linked-Reads

2017 ◽  
Author(s):  
Patrick Marks ◽  
Sarah Garcia ◽  
Alvaro Martinez Barrio ◽  
Kamila Belhocine ◽  
Jorge Bernate ◽  
...  
Keyword(s):  
Human Genome ◽  
Large Scale ◽  
De Novo ◽  
Simultaneous Detection ◽  
Whole Genome ◽  
Structural Variations ◽  
Full Spectrum ◽  
Short Read ◽  
Short Reads ◽  
A Genome

AbstractLarge-scale population based analyses coupled with advances in technology have demonstrated that the human genome is more diverse than originally thought. To date, this diversity has largely been uncovered using short read whole genome sequencing. However, standard short-read approaches, used primarily due to accuracy, throughput and costs, fail to give a complete picture of a genome. They struggle to identify large, balanced structural events, cannot access repetitive regions of the genome and fail to resolve the human genome into its two haplotypes. Here we describe an approach that retains long range information while harnessing the advantages of short reads. Starting from only ∼1ng of DNA, we produce barcoded short read libraries. The use of novel informatic approaches allows for the barcoded short reads to be associated with the long molecules of origin producing a novel datatype known as ‘Linked-Reads’. This approach allows for simultaneous detection of small and large variants from a single Linked-Read library. We have previously demonstrated the utility of whole genome Linked-Reads (lrWGS) for performing diploid, de novo assembly of individual genomes (Weisenfeld et al. 2017). In this manuscript, we show the advantages of Linked-Reads over standard short read approaches for reference based analysis. We demonstrate the ability of Linked-Reads to reconstruct megabase scale haplotypes and to recover parts of the genome that are typically inaccessible to short reads, including phenotypically important genes such as STRC, SMN1 and SMN2. We demonstrate the ability of both lrWGS and Linked-Read Whole Exome Sequencing (lrWES) to identify complex structural variations, including balanced events, single exon deletions, and single exon duplications. The data presented here show that Linked-Reads provide a scalable approach for comprehensive genome analysis that is not possible using short reads alone.

scholarly journals Refined detection and phasing of structural aberrations in pediatric acute lymphoblastic leukemia by linked-read whole genome sequencing

2018 ◽  
Author(s):  
Jessica Nordlund ◽  
Yanara Marincevic-Zuniga ◽  
Lucia Cavelier ◽  
Amanda Raine ◽  
Tom Martin ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Lymphoblastic Leukemia ◽  
Chromosomal Rearrangements ◽  
Whole Genome ◽  
Structural Variants ◽  
Short Read ◽  
Pediatric Acute Lymphoblastic Leukemia ◽  
Dna Quality

ABSTRACTStructural chromosomal rearrangements that may lead to in-frame gene-fusions represent a leading source of information for diagnosis, risk stratification, and prognosis in pediatric acute lymphoblastic leukemia (ALL). However, short-read whole genome sequencing (WGS) technologies struggle to accurately identify and phase such large-scale chromosomal aberrations in cancer genomes. We therefore evaluated linked-read WGS for detection of chromosomal rearrangements in an ALL cell line (REH) and primary samples of varying DNA quality from 12 patients diagnosed with ALL. We assessed the effect of input DNA quality on phased haplotype block size and the detectability of copy number aberrations (CNAs) and structural variants (SVs). Biobanked DNA isolated by standard column-based extraction methods was sufficient to detect chromosomal rearrangements even at low 10x sequencing coverage. Linked-read WGS enabled precise, allele-specific, digital karyotyping at a base-pair resolution for a wide range of structural variants including complex rearrangements and aneuploidy assessment. With use of haplotype information from the linked-reads, we also identified additional structural variants, such as a compound heterozygous deletion of ERG in a patient with the DUX4-IGH fusion gene. Thus, linked-read WGS allows detection of important pathogenic variants in ALL genomes at a resolution beyond that of traditional karyotyping or short-read WGS.

scholarly journals Detection and assembly of novel sequence insertions using Linked-Read technology

2019 ◽  
Author(s):  
Dmitry Meleshko ◽  
Patrick Marks ◽  
Stephen Williams ◽  
Iman Hajirasouliha
Keyword(s):  
Dna Sequences ◽  
De Novo Assembly ◽  
De Novo ◽  
Supplementary Information ◽  
Computational Techniques ◽  
Whole Genome ◽  
Structural Variations ◽  
Short Read ◽  
Link Type ◽  
Long Read

AbstractMotivationEmerging Linked-Read (aka read-cloud) technologies such as the 10x Genomics Chromium system have great potential for accurate detection and phasing of largescale human genome structural variations (SVs). By leveraging the long-range information encoded in Linked-Read sequencing, computational techniques are able to detect and characterize complex structural variations that are previously undetectable by short-read methods. However, there is no available Linked-Read method for detection and assembly of novel sequence insertions, DNA sequences present in a given sequenced sample but missing in the reference genome, without requiring whole genome de novo assembly. In this paper, we propose a novel integrated alignment-based and local-assembly-based algorithm, Novel-X, that effectively uses the barcode information encoded in Linked-Read sequencing datasets to improve detection of such events without the need of whole genome de novo assembly. We evaluated our method on two haploid human genomes, CHM1 and CHM13, sequenced on the 10x Genomics Chromium system. These genomes have been also characterized with high coverage PacBio long-reads recently. We also tested our method on NA12878, the wellknown HapMap CEPH diploid genome and the child genome in a Yoruba trio (NA19240) which was recently studied on multiple sequencing platforms. Detecting insertion events is very challenging using short reads and the only viable available solution is by long-read sequencing (e.g. PabBio or ONT). Our experiments, however, show that Novel-X finds many insertions that cannot be found by state of the art tools using short-read sequencing data but present in PacBio data. Since Linked-Read sequencing is significantly cheaper than long-read sequencing, our method using Linked-Reads enables routine large-scale screenings of sequenced genomes for novel sequence insertions.AvailabilitySoftware is freely available at https://github.com/1dayac/[email protected] informationSupplementary data are available at https://github.com/1dayac/novel_insertions_supplementary

scholarly journals A study of transposable element-associated structural variations (TASVs) using a de novo-assembled Korean genome

2021 ◽  
Author(s):  
Seyoung Mun ◽  
Songmi Kim ◽  
Wooseok Lee ◽  
Keunsoo Kang ◽  
Thomas J. Meyer ◽  
...  
Keyword(s):  
Genome Sequencing ◽  
Genome Assembly ◽  
De Novo ◽  
Personal Genome ◽  
Human Populations ◽  
Whole Genome ◽  
Structural Variations ◽  
Insert Size ◽  
Human Genomes ◽  
Next Generation Sequencing Ngs

AbstractAdvances in next-generation sequencing (NGS) technology have made personal genome sequencing possible, and indeed, many individual human genomes have now been sequenced. Comparisons of these individual genomes have revealed substantial genomic differences between human populations as well as between individuals from closely related ethnic groups. Transposable elements (TEs) are known to be one of the major sources of these variations and act through various mechanisms, including de novo insertion, insertion-mediated deletion, and TE–TE recombination-mediated deletion. In this study, we carried out de novo whole-genome sequencing of one Korean individual (KPGP9) via multiple insert-size libraries. The de novo whole-genome assembly resulted in 31,305 scaffolds with a scaffold N50 size of 13.23 Mb. Furthermore, through computational data analysis and experimental verification, we revealed that 182 TE-associated structural variation (TASV) insertions and 89 TASV deletions contributed 64,232 bp in sequence gain and 82,772 bp in sequence loss, respectively, in the KPGP9 genome relative to the hg19 reference genome. We also verified structural differences associated with TASVs by comparative analysis with TASVs in recent genomes (AK1 and TCGA genomes) and reported their details. Here, we constructed a new Korean de novo whole-genome assembly and provide the first study, to our knowledge, focused on the identification of TASVs in an individual Korean genome. Our findings again highlight the role of TEs as a major driver of structural variations in human individual genomes.

scholarly journals Unravelling the Sequential Interplay of Mutational Mechanisms during Clonal Evolution in Relapsed Pediatric Acute Lymphoblastic Leukemia

Genes ◽  
2021 ◽  
Vol 12 (2) ◽  
pp. 214
Author(s):  
Željko Antić ◽  
Stefan H. Lelieveld ◽  
Cédric G. van der Ham ◽  
Edwin Sonneveld ◽  
Peter M. Hoogerbrugge ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
De Novo ◽  
Lymphoblastic Leukemia ◽  
Clonal Evolution ◽  
Leukemic Cells ◽  
Proliferative Potential ◽  
Disease Evolution ◽  
Pediatric Acute Lymphoblastic Leukemia ◽  
First Relapse ◽  
Mutational Processes

Pediatric acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy and is characterized by clonal heterogeneity. Genomic mutations can increase proliferative potential of leukemic cells and cause treatment resistance. However, mechanisms driving mutagenesis and clonal diversification in ALL are not fully understood. In this proof of principle study, we performed whole genome sequencing of two cases with multiple relapses in order to investigate whether groups of mutations separated in time show distinct mutational signatures. Based on mutation allele frequencies at diagnosis and subsequent relapses, we clustered mutations into groups and performed cluster-specific mutational profile analysis and de novo signature extraction. In patient 1, who experienced two relapses, the analysis unraveled a continuous interplay of aberrant activation induced cytidine deaminase (AID)/apolipoprotein B editing complex (APOBEC) activity. The associated signatures SBS2 and SBS13 were present already at diagnosis, and although emerging mutations were lost in later relapses, the process remained active throughout disease evolution. Patient 2 had three relapses. We identified episodic mutational processes at diagnosis and first relapse leading to mutations resembling ultraviolet light-driven DNA damage, and thiopurine-associated damage at first relapse. In conclusion, our data shows that investigation of mutational processes in clusters separated in time may aid in understanding the mutational mechanisms and discovery of underlying causes.

scholarly journals P190BCR-ABL1 in a Patient with Philadelphia Chromosome Positive T-Cell Acute Lymphoblastic Leukemia: A Rare Case Report and Review of Literature

2021 ◽  
pp. 1040-1050
Author(s):  
Samah Kohla ◽  
Sarah EL Kourashy ◽  
Zafar Nawaz ◽  
Reda Youssef ◽  
Ahmad Al-Sabbagh ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Rare Case ◽  
De Novo ◽  
Lymphoblastic Leukemia ◽  
Prognostic Significance ◽  
Philadelphia Chromosome ◽  
Cytogenetic Abnormality ◽  
Rare Case Report ◽  
Philadelphia Chromosome Positive

T-acute lymphoblastic leukemia/lymphoblastic lymphoma (T-ALL/LBL) is rare and aggressive leukemia. Philadelphia chromosome positive (Ph+) is the most common cytogenetic abnormality in chronic myeloid leukemia (CML) and B-acute lymphoblastic leukemia (B-ALL). Ph+ T-ALL is exceeding rare and has a therapeutic and prognostic significance. The incidence and outcome of Ph+ T-ALL are unknown. Differentiation between Ph+ T-ALL/LBL and T-cell lymphoblastic crises of CML may be difficult. We report a rare case of adult de novo T-ALL with significant monocytosis, having Ph+ with (P190 <i>BCR-ABL1</i>) as a cytogenetic abnormality. He was treated with ALL induction chemotherapy and imatinib and achieved complete remission, then relapsed twice and expired shortly after the last CNS relapse.

scholarly journals MUM&Co: accurate detection of all SV types through whole-genome alignment

2020 ◽  
Vol 36 (10) ◽  
pp. 3242-3243 ◽  
Author(s):  
Samuel O’Donnell ◽  
Gilles Fischer
Keyword(s):  
De Novo ◽  
Supplementary Information ◽  
Genome Alignment ◽  
Whole Genome ◽  
Structural Variations ◽  
Sequencing Technologies ◽  
Third Generation Sequencing ◽  
Human Genomes ◽  
Whole Genome Alignment ◽  
Primary Output

Abstract Summary MUM&Co is a single bash script to detect structural variations (SVs) utilizing whole-genome alignment (WGA). Using MUMmer’s nucmer alignment, MUM&Co can detect insertions, deletions, tandem duplications, inversions and translocations greater than 50 bp. Its versatility depends upon the WGA and therefore benefits from contiguous de-novo assemblies generated by third generation sequencing technologies. Benchmarked against five WGA SV-calling tools, MUM&Co outperforms all tools on simulated SVs in yeast, plant and human genomes and performs similarly in two real human datasets. Additionally, MUM&Co is particularly unique in its ability to find inversions in both simulated and real datasets. Lastly, MUM&Co’s primary output is an intuitive tabulated file containing a list of SVs with only necessary genomic details. Availability and implementation https://github.com/SAMtoBAM/MUMandCo. Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals High incidence of disseminated intravascular coagulation during remission induction of adult patients with acute lymphoblastic leukemia [see comments]

Blood ◽  
1992 ◽  
Vol 79 (5) ◽  
pp. 1305-1310 ◽  
Author(s):  
AH Sarris ◽  
S Kempin ◽  
E Berman ◽  
J Michaeli ◽  
C Little ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Disseminated Intravascular Coagulation ◽  
De Novo ◽  
Sinus Thrombosis ◽  
Lymphoblastic Leukemia ◽  
Philadelphia Chromosome ◽  
Temporal Association ◽  
Remission Induction ◽  
Time Interval ◽  
Intravascular Coagulation

Abstract We determined the incidence and complications of disseminated intravascular coagulation (DIC) at presentation and during remission induction of previously untreated adults with acute lymphoblastic leukemia (ALL) or de novo Philadelphia chromosome-positive ALL (PCALL) seen at Memorial Hospital between January 1, 1978 and December 31, 1989. DIC was diagnosed in the presence of (1) low fibrinogen (less than or equal to 160 mg/dL), (2) prolonged prothrombin time (PT) and falling fibrinogen, or (3) prolonged PT and positive fibrin split products (FSP). L-Asparaginase was not used during remission induction. Among adequately screened patients with ALL, DIC was detected in 7 of 58 (12%) before initiation of chemotherapy and in 35 of 45 (78%) during remission induction. DIC was not simply the result of infection because clinical and laboratory signs of infection were absent in 16 patients, whereas only 2 of the 22 febrile patients with DIC had positive cultures. Among the 38 patients with DIC at presentation or during remission induction, serious complications were seen in 13 in temporal association with DIC (pulmonary embolus in one, sagittal sinus thrombosis in three, and serious hemorrhage in nine) and were major factors in the deaths of three patients. Among the 10 patients with thorough screening but no evidence of DIC there was only one hemorrhage during the same time interval. In patients with PCALL, DIC was detected in 9% at presentation and in 80% during remission induction. We conclude that DIC is rare at presentation but common during remission induction of adult ALL and PCALL and may be associated with significant thrombotic and hemorrhagic complications. We suggest daily screening for DIC during the first 14 days of remission induction. The treatment of DIC in ALL and PCALL should be a subject of future clinical studies.

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