June 1, 2021  |  

Single Molecule Real Time (SMRT) sequencing sensitively detects polyclonal and compound BCR-ABL in patients who relapse on kinase inhibitor therapy.

Secondary kinase domain (KD) mutations are the most well-recognized mechanism of resistance to tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML) and other cancers. In some cases, multiple drug resistant KD mutations can coexist in an individual patient (“polyclonality”). Alternatively, more than one mutation can occur in tandem on a single allele (“compound mutations”) following response and relapse to sequentially administered TKI therapy. Distinguishing between these two scenarios can inform the clinical choice of subsequent TKI treatment. There is currently no clinically adaptable methodology that offers the ability to distinguish polyclonal from compound mutations. Due to the size of the BCR-ABL KD where TKI-resistant mutations are detected, next-generation platforms are unable to generate reads of sufficient length to determine if two mutations separated by 500 nucleotides reside on the same allele. Pacific Biosciences RS Single Molecule Real-Time (SMRT) circular consensus sequencing technology is a novel third generation deep sequencing technology capable of rapidly and reliably achieving average read lengths of ~1000 bp and frequently beyond 3000 bp, allowing sequencing of the entire ABL KD on single strand of DNA. We sought to address the ability of SMRT sequencing technology to distinguish polyclonal from compound mutations using clinical samples obtained from patients who have relapsed on BCR-ABL TKI treatment.


June 1, 2021  |  

Allelic specificity of immunoglobulin heavy chain ([email protected]) translocation in B-cell acute lymphoblastic leukemia (B-ALL) unveiled by long-read sequencing

Oncogenic fusion of IGH-DUX4 has recently been reported as a hallmark that defines a B-ALL subtype present in up to 7% of adolescents and young adults B-ALL. The translocation of DUX4 into IGH results in aberrant activation of DUX4 by hijacking the intronic IGH enhancer (Eµ). How IGH-DUX4 translocation interplays with IGH allelic exclusion was never been explored. We investigated this in Nalm6 B-ALL cell line, using long-read (PacBio Iso-Seq method and 10X Chromium WGS), short-read (Illumina total stranded RNA and WGS), epigenome (H3K27ac ChIP-seq, ATAC-seq) and 3-D genome (Hi-C, H3K27ac HiChIP, Capture-C).


April 21, 2020  |  

Chromosome-length haplotigs for yak and cattle from trio binning assembly of an F1 hybrid

Background Assemblies of diploid genomes are generally unphased, pseudo-haploid representations that do not correctly reconstruct the two parental haplotypes present in the individual sequenced. Instead, the assembly alternates between parental haplotypes and may contain duplications in regions where the parental haplotypes are sufficiently different. Trio binning is an approach to genome assembly that uses short reads from both parents to classify long reads from the offspring according to maternal or paternal haplotype origin, and is thus helped rather than impeded by heterozygosity. Using this approach, it is possible to derive two assemblies from an individual, accurately representing both parental contributions in their entirety with higher continuity and accuracy than is possible with other methods.Results We used trio binning to assemble reference genomes for two species from a single individual using an interspecies cross of yak (Bos grunniens) and cattle (Bos taurus). The high heterozygosity inherent to interspecies hybrids allowed us to confidently assign >99% of long reads from the F1 offspring to parental bins using unique k-mers from parental short reads. Both the maternal (yak) and paternal (cattle) assemblies contain over one third of the acrocentric chromosomes, including the two largest chromosomes, in single haplotigs.Conclusions These haplotigs are the first vertebrate chromosome arms to be assembled gap-free and fully phased, and the first time assemblies for two species have been created from a single individual. Both assemblies are the most continuous currently available for non-model vertebrates.MbmegabaseskbkilobasesMYAmillions of years agoMHCmajor histocompatibility complexSMRTsingle molecule real time


April 21, 2020  |  

Acquired N-Linked Glycosylation Motifs in B-Cell Receptors of Primary Cutaneous B-Cell Lymphoma and the Normal B-Cell Repertoire.

Primary cutaneous follicle center lymphoma (PCFCL) is a rare mature B-cell lymphoma with an unknown etiology. PCFCL resembles follicular lymphoma (FL) by cytomorphologic and microarchitectural criteria. FL B cells are selected for N-linked glycosylation motifs in their B-cell receptors (BCRs) that are acquired during continuous somatic hypermutation. The stimulation of mannosylated BCR by lectins on the tumor microenvironment is therefore a candidate driver in FL pathogenesis. We investigated whether the same mechanism could play a role in PCFCL pathogenesis. Full-length functional variable, diversity, and joining gene sequences of 18 PCFCL and 8 primary cutaneous diffuse large B-cell lymphoma, leg-type were identified by unbiased Anchoring Reverse Transcription of Immunoglobulin Sequences and Amplification by Nested PCR and BCR reconstruction from RNA sequencing data. Low BCR variation demonstrated negligible ongoing somatic hypermutation in PCFCL and primary cutaneous diffuse large B-cell lymphoma, leg-type, and indicated that the PCFCL microarchitecture does not act as a functional germinal center. Similar to FL but in contrast to primary cutaneous diffuse large B-cell lymphoma, leg-type, BCR genes of 15 PCFCLs (83%) had acquired N-linked glycosylation motifs. These motifs were located at the BCR positions converted to N-linked glycosylation motifs in normal B-cell repertoires with low prevalence but mostly at different positions than those found in FL. The cutaneous localization of PCFCL might suggest a role for lectins from commensal skin bacteria in PCFCL lymphomagenesis.Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.


April 21, 2020  |  

Mogamulizumab Treatment Elicits Autoantibodies Attacking the Skin in Patients with Adult T-Cell Leukemia-Lymphoma.

Purpose: The anti-CCR4 mAb, mogamulizumab, offers therapeutic benefit to patients with adult T-cell leukemia-lymphoma (ATL), but skin-related adverse events (AE) such as erythema multiforme occur frequently. The purpose of this study was to determine the mechanisms by which mogamulizumab causes skin-related AEs in patients with ATL.Experimental Design: We investigated whether autoantibodies were present in patients’ sera using flow cytometry to determine binding to keratinocytes and melanocytes (n = 17), and immunofluorescence analysis of tissue sections. We analyzed the IgM heavy chain repertoire in peripheral blood mononuclear cells before and after mogamulizumab or other chemotherapy by next-generation sequencing (NGS; n = 16).Results: Autoantibodies recognizing human keratinocytes or melanocytes were found in the sera of 6 of 8 patients suffering from mogamulizumab-induced erythema multiforme. In one patient, complement-dependent cytotoxicity (CDC) mediated by autoantibodies against keratinocytes or melanocytes was proportionally related to the severity of the erythema multiforme. The presence of autoantibodies in the epidermis was confirmed in all biopsy specimens of mogamulizumab-induced erythema multiforme (n = 12). Furthermore, colocalization of autoantibodies and C1q, suggesting the activation of CDC, was observed in 67% (8/12). In contrast, no autoantibody or C1q was found in ATL tumor skin lesions (n = 13). Consistent with these findings, NGS demonstrated that IgM germline genes had newly emerged and expanded, resulting in IgM repertoire skewing at the time of erythema multiforme.Conclusions: Mogamulizumab elicits autoantibodies playing an important role in skin-related AEs, possibly associated with regulatory T-cell depletion. This is the first report demonstrating the presence of skin-directed autoantibodies after mogamulizumab treatment. ©2019 American Association for Cancer Research.


April 21, 2020  |  

Consensus and variations in cell line specificity among human metapneumovirus strains.

Human metapneumovirus (HMPV) has been a notable etiological agent of acute respiratory infection in humans, but it was not discovered until 2001, because HMPV replicates only in a limited number of cell lines and the cytopathic effect (CPE) is often mild. To promote the study of HMPV, several groups have generated green fluorescent protein (GFP)-expressing recombinant HMPV strains (HMPVGFP). However, the growing evidence has complicated the understanding of cell line specificity of HMPV, because it seems to vary notably among HMPV strains. In addition, unique A2b clade HMPV strains with a 180-nucleotide duplication in the G gene (HMPV A2b180nt-dup strains) have recently been detected. In this study, we re-evaluated and compared the cell line specificity of clinical isolates of HMPV strains, including the novel HMPV A2b180nt-dup strains, and six recombinant HMPVGFP strains, including the newly generated recombinant HMPV A2b180nt-dup strain, MG0256-EGFP. Our data demonstrate that VeroE6 and LLC-MK2 cells generally showed the highest infectivity with any clinical isolates and recombinant HMPVGFP strains. Other human-derived cell lines (BEAS-2B, A549, HEK293, MNT-1, and HeLa cells) showed certain levels of infectivity with HMPV, but these were significantly lower than those of VeroE6 and LLC-MK2 cells. Also, the infectivity in these suboptimal cell lines varied greatly among HMPV strains. The variations were not directly related to HMPV genotypes, cell lines used for isolation and propagation, specific genome mutations, or nucleotide duplications in the G gene. Thus, these variations in suboptimal cell lines are likely intrinsic to particular HMPV strains.


April 21, 2020  |  

Single-Molecule Sequencing: Towards Clinical Applications.

In the past several years, single-molecule sequencing platforms, such as those by Pacific Biosciences and Oxford Nanopore Technologies, have become available to researchers and are currently being tested for clinical applications. They offer exceptionally long reads that permit direct sequencing through regions of the genome inaccessible or difficult to analyze by short-read platforms. This includes disease-causing long repetitive elements, extreme GC content regions, and complex gene loci. Similarly, these platforms enable structural variation characterization at previously unparalleled resolution and direct detection of epigenetic marks in native DNA. Here, we review how these technologies are opening up new clinical avenues that are being applied to pathogenic microorganisms and viruses, constitutional disorders, pharmacogenomics, cancer, and more.Copyright © 2018 Elsevier Ltd. All rights reserved.


April 21, 2020  |  

Long-read sequencing unveils IGH-DUX4 translocation into the silenced IGH allele in B-cell acute lymphoblastic leukemia.

[email protected] proto-oncogene translocation is a common oncogenic event in lymphoid lineage cancers such as B-ALL, lymphoma and multiple myeloma. Here, to investigate the interplay between [email protected] proto-oncogene translocation and IGH allelic exclusion, we perform long-read whole-genome and transcriptome sequencing along with epigenetic and 3D genome profiling of Nalm6, an IGH-DUX4 positive B-ALL cell line. We detect significant allelic imbalance on the wild-type over the IGH-DUX4 haplotype in expression and epigenetic data, showing IGH-DUX4 translocation occurs on the silenced IGH allele. In vitro, this reduces the oncogenic stress of DUX4 high-level expression. Moreover, patient samples of IGH-DUX4 B-ALL have similar expression profile and IGH breakpoints as Nalm6, suggesting a common mechanism to allow optimal dosage of non-toxic DUX4 expression.


April 21, 2020  |  

Sequence properties of certain GC rich avian genes, their origins and absence from genome assemblies: case studies.

More and more eukaryotic genomes are sequenced and assembled, most of them presented as a complete model in which missing chromosomal regions are filled by Ns and where a few chromosomes may be lacking. Avian genomes often contain sequences with high GC content, which has been hypothesized to be at the origin of many missing sequences in these genomes. We investigated features of these missing sequences to discover why some may not have been integrated into genomic libraries and/or sequenced.The sequences of five red jungle fowl cDNA models with high GC content were used as queries to search publicly available datasets of Illumina and Pacbio sequencing reads. These were used to reconstruct the leptin, TNFa, MRPL52, PCP2 and PET100 genes, all of which are absent from the red jungle fowl genome model. These gene sequences displayed elevated GC contents, had intron sizes that were sometimes larger than non-avian orthologues, and had non-coding regions that contained numerous tandem and inverted repeat sequences with motifs able to assemble into stable G-quadruplexes and intrastrand dyadic structures. Our results suggest that Illumina technology was unable to sequence the non-coding regions of these genes. On the other hand, PacBio technology was able to sequence these regions, but with dramatically lower efficiency than would typically be expected.High GC content was not the principal reason why numerous GC-rich regions of avian genomes are missing from genome assembly models. Instead, it is the presence of tandem repeats containing motifs capable of assembling into very stable secondary structures that is likely responsible.


April 21, 2020  |  

Single-molecule sequencing detection of N6-methyladenine in microbial reference materials.

The DNA base modification N6-methyladenine (m6A) is involved in many pathways related to the survival of bacteria and their interactions with hosts. Nanopore sequencing offers a new, portable method to detect base modifications. Here, we show that a neural network can improve m6A detection at trained sequence contexts compared to previously published methods using deviations between measured and expected current values as each adenine travels through a pore. The model, implemented as the mCaller software package, can be extended to detect known or confirm suspected methyltransferase target motifs based on predictions of methylation at untrained contexts. We use PacBio, Oxford Nanopore, methylated DNA immunoprecipitation sequencing (MeDIP-seq), and whole-genome bisulfite sequencing data to generate and orthogonally validate methylomes for eight microbial reference species. These well-characterized microbial references can serve as controls in the development and evaluation of future methods for the identification of base modifications from single-molecule sequencing data.


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