Sequencing Acute Myeloid Leukemia Genomes with “Next Generation” Technologies.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. sci-36-sci-36
Author(s):  
Elaine Mardis ◽  
Timothy J. Ley ◽  
Richard K. Wilson
Keyword(s):  
Whole Genome Sequencing ◽  
Tumor Cells ◽  
Genome Sequencing ◽  
Genomic Dna ◽  
Myeloid Leukemia ◽  
Somatic Mutations ◽  
Normal Skin ◽  
Whole Genome ◽  
The Novel ◽  
Tumor Genome

Abstract For most patients with a sporadic presentation of acute myeloid leukemia (AML), neither the initiating nor the progression mutations responsible for disease are known. Recent attempts to identify key mutations with directed sequencing approaches, or with array-based genomic studies, have had limited success, suggesting that unbiased whole genome sequencing approaches may be required to identify most of the mutations responsible for AML pathogenesis. Until recently, whole genome sequencing has been impractical due to the high cost of conventional capillary-based sequencing and the large numbers of enriched primary tumor cells required to yield the necessary genomic DNA for library preparation. “Next Generation” sequencing approaches have changed this landscape dramatically. Using the Solexa/Illumina platform, we have now sequenced the genomic DNA of highly enriched tumor cells and normal skin cells obtained from a carefully selected patient with a typical presentation of FAB M1 AML. We obtained 98.2 billion bases of sequences from the cytogenetically normal tumor cell genome (32.7 fold haploid coverage), and 41.8 billion bases of sequence from the normal skin genome (13.9 fold coverage). Using these data, we detected diploid sequence coverage of 91% of 46,320 heterozygous SNPs, defined in the tumor genome (by array-based genotyping), and 83% diploid coverage of the skin genome. Of 2,647,695 well-supported single nucleotide variants in the tumor genome, 2,588,486 (97.7%) were also detected in the patient’s skin genome, defining them as inherited. From the remaining variants, 8 have been fully validated as somatic mutations by conventional capillary sequencing using PCR-generated amplicons. We also detected somatic mutations in the FLT3 (ITD) and NPM1 genes (a classic NPMc mutation). Based on deep read-count data of the novel variants on a 454 sequencer, we hypothesize that all of the mutations are in virtually all of the tumor cells, and all were retained at relapse 11 months later, suggesting that a single dominant clone contained all of the mutations. None of the novel mutations has previously been detected in AML cases (and none were found in any of 187 additional AML cases studied here). A number of additional potential somatic mutations in regions lying near genes (but not altering coding sequences) are currently being validated and tested for recurrence in other AML samples. Whole genome sequencing of a second M1 AML genome is now underway. These results demonstrate the power of unbiased whole genome sequencing approaches to discover cancer-associated mutations in novel candidate genes.

Whole Genome Sequencing Reveals Novel Recurring Somatic Mutations Affecting HUWE1 and DIAPH2 Genes in Multiple Myeloma

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 320-320
Author(s):  
Michael H. Tomasson ◽  
Dong Shen ◽  
Vishwanathan Hucthagowder ◽  
William Schierding ◽  
Chelsea D. Mullins ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
B Cells ◽  
Whole Genome Sequencing ◽  
Tumor Cells ◽  
Genome Sequencing ◽  
Peripheral Blood ◽  
Somatic Mutations ◽  
Chromosomal Translocations ◽  
Whole Genome ◽  
Normal Controls

Abstract Abstract 320 Multiple myeloma (MM) is an incurable malignancy of antibody secreting plasma B-cells whose etiology is still poorly understood. We used whole genome sequencing (WGS) and deep-read capture validation to thoroughly characterize the mutation landscape in four newly diagnosed MM patients and further examined mutation recurrence in 89 additional MM patients. WGS cases were selected to be racially diverse and represent both hyperdiploid and non-hyperdiploid MM with otherwise “simple” karyotypes. All studies to date have used peripheral blood as controls, however, abnormal B cells and circulating tumor cells frequently contaminate the peripheral blood of MM patients and these studies may have missed the early genetic events potentially important in disease pathogenesis. We therefore chose to use matched skin samples as normal controls. The use of skin DNA controls and deep read count capture validation of single nucleotide variants (SNV) allowed us, for the first time, to demonstrate clear separation between normal and malignant cell populations. Our analysis pipeline detected both somatic SNVs and structural variants (SV, ie. translocations, deletions, insertions). In all four patients, we observed chromosomal translocations at the Ig heavy chain locus, VDJ recombination, and Ig locus somatic hypermuation. We found somatic mutations affecting the E3 ubiquitin ligase HUWE1 in 4% (4/94) of cases, as well as recurring deletions affecting the Rho pathway regulator DIAPH2 (recurrence data will be presented). RNA sequencing was preformed for 3 of the WGS patients to explore transcriptional effects of mutations observed. Statistical analysis identified significantly mutated genes, i.e. mutation hotspots, including ROBO2, BCL6 and cadherin/catenin genes. Genes involved in cell polarity, cell adhesion and axon guidance were affected in all four WGS patients. Chromosomal translocations present in all four WGS patients involved both known oncogenes (CCND1, MYC and MAFB) and putative oncogenes. A novel translocation implicated KCNT2, encoding a sodium-activated potassium channel, as a potential oncogene in MM. Recurrent point mutations were relatively rare in MM, suggesting that SVs are more common driver mutations and/or that SNVs in MM disrupt a diverse array of genes to affect key pathways. For genomic studies in MM moving forward, our results emphasize the importance of matched normal controls uncontaminated by tumor cells, and suggest that careful analysis of SVs be included to find novel oncogenes and important clinical correlations. Disclosures: No relevant conflicts of interest to declare.

Whole Genome Sequencing of Therapy-Related Acute Myeloid Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 784-784
Author(s):  
Giridharan Ramsingh ◽  
Dong Shen ◽  
Tamara Lamprecht ◽  
Sharon Heath ◽  
Robert S. Fulton ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Myeloid Leukemia ◽  
Somatic Mutations ◽  
De Novo ◽  
Whole Genome ◽  
Hematopoietic Stem ◽  
De Novo Aml ◽  
Acute Myeloid

Abstract Abstract 784 Whole Genome Sequencing of Therapy-Related Acute Myeloid Leukemia Giridharan Ramsingh, Dong Shen, Tamara L. Lamprecht, Sharon E. Heath, Robert S. Fulton, Elaine Mardis, Li Ding, Peter Westervelt, John Welch, Matthew J. Walter, Timothy A. Graubert, John F. DiPersio, Timothy J. Ley, Richard K. Wilson, and Daniel C. Link. Therapy related therapy-related acute myeloid leukemia (t-AML) accounts for 10–20% of all new cases of AML, and its incidence is rising. A fundamental difference in the pathogenesis of de novo AML and t-AML is prior treatment with chemotherapy and/or radiotherapy. The exposure of hematopoietic stem/progenitors cells (HSPCs) to this genotoxic stress is hypothesized to alter the number and spectrum of mutations that arise in t-AML. Moreover, the genotoxic stress may exert selective pressure to expand those HSPC clones that are inherently resistant to chemotherapy, a common feature in t-AML. To test these hypotheses, we sequenced the genomes of 23 cases of t-AML and compared them to the genomes of 24 cases of de novo AML, which we recently reported (Welch et al., Cell, July 2012). We choose to focus our initial studies on the subset of t-AML with normal cytogenetics or simple balanced translocations. Specifically, MLL gene rearrangements were observed in 22% of cases, other balanced translocations in 22%, trisomy 8 in 22%, normal karyotype in 31%, and a complex karyotype in a single case. All patients had received prior alkylator chemotherapy (62%), topoisomerase inhibitor chemotherapy (65%), or radiotherapy (77%). To identify somatic mutations, whole genome sequencing was performed on leukemic bone marrow (average 65% blasts) and skin (normal) DNA. Average haploid coverage was 37.5X and 34.7X for the leukemia and skin genomes, respectively. All somatic mutations were verified using patient-specific custom NimbleGen capture arrays, followed by Illumina sequencing. Although the total number of somatic single nucleotide variants in older patients (>50 years) with t-AML was similar to that observed in de novo AML (484 ± 68 vs. 506 ± 45, respectively), significantly more mutations were present in younger (≤ 50 years) patients with t-AML (743 ± 228) compared with de novo AML (336 ± 179, P=0.04). Exposure to chemotherapy is associated with an increased rate of transversions in relapsed AML (Ding et al., Nature 2012). However, the percentage of somatic mutations that were transversions in t-AML (35.8 ± 1.91%) was similar to that seen in de novo AML (33.5 ± 0.93%), regardless of age. In the 23 t-AML genomes, we identified recurring mutations (present in at least 2 cases) in 20 genes. Many of these mutations were also observed in de novo AML genomes (Figure 1). The most commonly mutated gene in t-AML was TET2, which was mutated in 35% of cases. Of interest, missense mutations of the ABC transporter gene ABCG2 were significantly enriched in t-AML (2/23, 8.7%) compared with de novo AML (0 in 200 cases, P=0.01). ABCG2 (also known as breast cancer resistance protein, BCRP) has been implicated in chemotherapy resistance. ABCG2 is expressed at high levels in hematopoietic stem cells, where it is known to function as a key drug transporter. Studies are underway to define the frequency of ABCG2 mutations (and other ABC transporter genes) in a larger cohort of t-AML, including cases with alterations in chromosome 5 or 7 or with complex cytogenetic abnormalities. In summary, in younger patients with t-AML, the mutational burden is higher than that of de novo AML patients, possibly reflecting prior exposure to chemoradiotherapy, though no increase in transversions was observed. Mutations of ABCG2 may contribute to chemotherapy resistance in a subset of t-AML. Figure 1. Recurring mutations in t-AML (n = 23) compared with de novo AML (n = 24). Figure 1. Recurring mutations in t-AML (n = 23) compared with de novo AML (n = 24). Disclosures: Ley: Washington University: Patents & Royalties.

scholarly journals Quantifying the influence of mutation detection on tumour subclonal reconstruction

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Lydia Y. Liu ◽  
Vinayak Bhandari ◽  
Adriana Salcedo ◽  
Shadrielle M. G. Espiritu ◽  
Quaid D. Morris ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Cancer Cells ◽  
Clinical Outcomes ◽  
Cancer Cell ◽  
Genome Sequencing ◽  
Somatic Mutations ◽  
Mutation Detection ◽  
Cell Populations ◽  
Whole Genome ◽  
Prostate Cancers

AbstractWhole-genome sequencing can be used to estimate subclonal populations in tumours and this intra-tumoural heterogeneity is linked to clinical outcomes. Many algorithms have been developed for subclonal reconstruction, but their variabilities and consistencies are largely unknown. We evaluate sixteen pipelines for reconstructing the evolutionary histories of 293 localized prostate cancers from single samples, and eighteen pipelines for the reconstruction of 10 tumours with multi-region sampling. We show that predictions of subclonal architecture and timing of somatic mutations vary extensively across pipelines. Pipelines show consistent types of biases, with those incorporating SomaticSniper and Battenberg preferentially predicting homogenous cancer cell populations and those using MuTect tending to predict multiple populations of cancer cells. Subclonal reconstructions using multi-region sampling confirm that single-sample reconstructions systematically underestimate intra-tumoural heterogeneity, predicting on average fewer than half of the cancer cell populations identified by multi-region sequencing. Overall, these biases suggest caution in interpreting specific architectures and subclonal variants.

Device for whole genome sequencing single circulating tumor cells from whole blood

Lab on a Chip ◽  
2019 ◽  
Vol 19 (19) ◽  
pp. 3168-3178 ◽  
Author(s):  
Ren Li ◽  
Fei Jia ◽  
Weikai Zhang ◽  
Fanghao Shi ◽  
Zhiguo Fang ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Genome Sequencing ◽  
Microfluidic Chip ◽  
Whole Blood ◽  
Whole Genome

To sequence single circulating tumor cells (CTCs) from whole blood, a microfluidic chip was developed to perform blood filtering/CTC enrichment/CTC sorting and in situ MDA for whole genome sequencing.

DNA Sequencing of a Murine Acute Promyelocytic Leukemia (APL) Genome Using Next Generation Technology.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3965-3965
Author(s):  
Lukas D. Wartman ◽  
Li Ding ◽  
David E. Larson ◽  
Michael D. McLellan ◽  
Heather Schmidt ◽  
...  
Keyword(s):  
Mouse Model ◽  
Dna Sequencing ◽  
Whole Genome Sequencing ◽  
Acute Promyelocytic Leukemia ◽  
Genome Sequencing ◽  
Somatic Mutations ◽  
Promyelocytic Leukemia ◽  
The Other ◽  
Whole Genome ◽  
Base Pairs

Abstract Abstract 3965 Poster Board III-901 We have recently established that whole genome sequencing is a valid, unbiased approach that can identify novel candidate mutations that may be important for AML pathogenesis (Ley et al Nature 2008, Mardis et al NEJM 2009). Acute promyelocytic leukemia (APL, FAB M3 AML) is a subtype of AML characterized by the t(15;17)(q22;q11.2) translocation that creates an oncogenic fusion gene, PML-RARA. Our laboratory has previously modeled APL in a mouse in an effort to understand the genetic events that lead to the disease. In our knockin mouse model, a human PML-RARA cDNA was targeted to the 5' untranslated region of the mouse cathepsin G gene on chromosome 14 (mCG-PR). The targeting vector was transfected into the RW-4 embryonic stem cell line, derived from a 129/SvJ mouse. The transfected RW-4 cells were injected into C57Bl/6 blastocysts, and chimeric offspring were bred to C57Bl/6 mice. F1 129/SvJ x C57Bl/6 mice were subsequently backcrossed onto the B6/Taconic background for 10 generations before establishing a tumor watch. About 60% of the mCG-PR mice in the Bl/6 background develop a disease that closely resembles APL only after a latent period of 7-18 months, suggesting that additional progression mutations are required for APL development. Array-based genomic techniques (expression array studies and high resolution CGH) have revealed some recurring genetic alterations that may be relevant for progression (i.e. an interstitial deletion of chromosome 2, trisomy 15, etc.), but gene-specific progression mutations have not yet been identified. To begin to identify these mutations in an unbiased fashion, we sequenced a cytogenetically normal, diploid mouse APL genome using massively parallel DNA sequencing via the Illumina platform. Since the tumor arose in a highly inbred mouse strain, we predicted that 15x coverage of the genome (approximately 40 billion base pairs of sequence) would be necessary to identify >90% of the heterozygous somatic mutations. We generated 2 Illumina paired-end libraries (insert sizes of 300-350 bp and 550-600 bp) and generated 59.64 billion base pairs of sequence with 3 full sequencing runs; the reads that successfully mapped generated 15.6x coverage. The sequence data predicted 87,778 heterozygous Single Nucleotide Variants (SNVs) compared to the mouse C57Bl6/J reference sequence, and 23,439 homozygous SNVs. Of the predicted heterozygous SNVs, 695 were non-synonymous (missense or nonsense, or altering a canonical splice site). Thus far, 80 of these putative non-synonymous SNVs have been further analyzed using Sanger sequencing of the original tumor DNA vs. pooled B6/Taconic spleen DNA and pooled129/SvJ spleen DNA as controls. 37/80 were shown to be false positive calls, and 37 were inherited SNPs from residual regions of the129/SvJ genome. 6/80 were present only in the tumor genome, and were candidate somatic mutations. These 6 were screened in 89 additional murine APL tumor samples derived from the same mouse model. Mutations in the Jarid2 (L915I) and Capns2 (N149S) genes occurred only in the proband, and are therefore of uncertain significance. 4/6 mutations were found in additional samples; 3 of these mutations were derived from a common ancestor of the proband and the other affected mice, and were therefore not relevant for pathogenesis. The other recurring mutation was in the pseudokinase domain of JAK1 (V657F), and was identified in one other mouse that was not closely related to the proband. This mutation is orthologous to the known activating mutation V617F in human JAK2, and is identical to a recently described JAK1 pseudokinase domain mutation (V658F) found in human APL and T-ALL samples (EG Jeong et al, Clin Can Res 14: 3716, 2008). We are currently testing the functional significance of this mutation by expressing it in bone marrow cells derived from young WT vs. mCG-PR mice. In summary, unbiased whole genome sequencing of a mouse APL genome has identified a recurring mutation of JAK1 found in both human and mouse APL samples. This approach may allow us to rapidly identify progression mutations that are common to human and murine AML, and provides an important proof-of-concept that this mouse model of AML is functionally related to its human counterpart. Disclosures: No relevant conflicts of interest to declare.

Complete Sequencing and Comparison of 12 Normal Karyotype M1 AML Genomes with 12 t(15;17) Positive M3-APL Genomes

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 404-404 ◽  
Author(s):  
John S. Welch ◽  
David Larson ◽  
Li Ding ◽  
Michael D. McLellan ◽  
Tamara Lamprecht ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Somatic Mutations ◽  
Normal Karyotype ◽  
Whole Genome ◽  
Single Mutation ◽  
Cohesin Complex ◽  
Diverse Range ◽  
Recurrent Mutations ◽  
Passenger Mutations

Abstract Abstract 404 To characterize the genomic events associated with distinct subtypes of AML, we used whole genome sequencing to compare 24 tumor/normal sample pairs from patients with normal karyotype (NK) M1-AML (12 cases) and t(15;17)-positive M3-AML (12 cases). All single nucleotide variants (SNVs), small insertions and deletions (indels), and cryptic structural variants (SVs) identified by whole genome sequencing (average coverage 28x) were validated using sample-specific custom Nimblegen capture arrays, followed by Illumina sequencing; an average coverage of 972 reads per somatic variant yielded 10,597 validated somatic variants (average 421/genome). Of these somatic mutations, 308 occurred in 286 unique genes; on average, 9.4 somatic mutations per genome had translational consequences. Several important themes emerged: 1) AML genomes contain a diverse range of recurrent mutations. We assessed the 286 mutated genes for recurrency in an additional 34 NK M1-AML cases and 9 M3-AML cases. We identified 51 recurrently mutated genes, including 37 that had not previously been described in AML; on average, each genome had 3 recurrently mutated genes (M1 = 3.2; M3 = 2.8, p = 0.32). 2) Many recurring mutations cluster in mutually exclusive pathways, suggesting pathophysiologic importance. The most commonly mutated genes were: FLT3 (36%), NPM1 (25%), DNMT3A (21%), IDH1 (18%), IDH2 (10%), TET2 (10%), ASXL1 (6%), NRAS (6%), TTN (6%), and WT1 (6%). In total, 3 genes (excluding PML-RARA) were mutated exclusively in M3 cases. 22 genes were found only in M1 cases (suggestive of alternative initiating mutations which occurred in methylation, signal transduction, and cohesin complex genes). 25 genes were mutated in both M1 and M3 genomes (suggestive of common progression mutations relevant for both subtypes). A single mutation in a cell growth/signaling gene occurred in 38 of 67 cases (FLT3, NRAS, RUNX1, KIT, CACNA1E, CADM2, CSMD1); these mutations were mutually exclusive of one another, and many of them occurred in genomes with PML-RARA, suggesting that they are progression mutations. We also identified a new leukemic pathway: mutations were observed in all four genes that encode members of the cohesin complex (STAG2, SMC1A, SMC3, RAD21), which is involved in mitotic checkpoints and chromatid separation. The cohesin mutations were mutually exclusive of each other, and collectively occur in 10% of non-M3 AML patients. 3) AML genomes also contain hundreds of benign “passenger” mutations. On average 412 somatic mutations per genome were translationally silent or occurred outside of annotated genes. Both M1 and M3 cases had similar total numbers of mutations per genome, similar mutation types (which favored C>T/G>A transitions), and a similar random distribution of variants throughout the genome (which was affected neither by coding regions nor expression levels). This is consistent with our recent observations of random “passenger” mutations in hematopoietic stem cell (HSC) clones derived from normal patients (Ley et al manuscript in preparation), and suggests that most AML-associated mutations are not pathologic, but pre-existed in the HSC at the time of initial transformation. In both studies, the total number of SNVs per genome correlated positively with the age of the patient (R2 = 0.48, p = 0.001), providing a possible explanation for the increasing incidence of AML in elderly patients. 4) NK M1 and M3 AML samples are mono- or oligo-clonal. By comparing the frequency of all somatic mutations within each sample, we could identify clusters of mutations with similar frequencies (leukemic clones) and determined that the average number of clones per genome was 1.8 (M1 = 1.5; M3 = 2.2; p = 0.04). 5) t(15;17) is resolved by a non-homologous end-joining repair pathway, since nucleotide resolution of all 12 t(15;17) breakpoints revealed inconsistent micro-homologies (0 – 7 bp). Summary: These data provide a genome-wide overview of NK and t(15;17) AML and provide important new insights into AML pathogenesis. AML genomes typically contain hundreds of random, non-genic mutations, but only a handful of recurring mutated genes that are likely to be pathogenic because they cluster in mutually exclusive pathways; specific combinations of recurring mutations, as well as rare and private mutations, shape the leukemia phenotype in an individual patient, and help to explain the clinical heterogeneity of this disease. Disclosures: Westervelt: Novartis: Speakers Bureau.

scholarly journals Whole-Genome Sequencing of a Human Clinical Isolate of the Novel Species Klebsiella quasivariicola sp. nov

2017 ◽  
Vol 5 (42) ◽  
Author(s):  
S. Wesley Long ◽  
Sarah E. Linson ◽  
Matthew Ojeda Saavedra ◽  
Concepcion Cantu ◽  
James J. Davis ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Clinical Isolate ◽  
Genome Sequencing ◽  
Novel Species ◽  
Whole Genome ◽  
The Novel ◽  
Short Read ◽  
Oxford Nanopore ◽  
Long Read

ABSTRACT In a study of 1,777 Klebsiella strains, we discovered KPN1705, which was distinct from all recognized Klebsiella spp. We closed the genome of strain KPN1705 using a hybrid of Illumina short-read and Oxford Nanopore long-read technologies. For this novel species, we propose the name Klebsiella quasivariicola sp. nov.

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