The omics era has greatly expanded the repertoire of approaches available for researchers and clinicians to unravel the complexity underpinning human health: Next Generation Sequencing (NGS) ...approaches can characterize genomes, epigenomes, transcriptomes and proteomes. The analyses are critical to assess in individuals both pre- and post-treatment during therapeutic development and early-stage clinical trials. Peripheral blood mononuclear cells (PBMCs) offer a non-invasive approach that, when combined with omics tools, can provide a near holistic view of immune processes across patient cohorts. Meanwhile, Formalin Fixed Paraffin Embedded (FFPE) tissues are a staple in clinical diagnostics and an ideal means to store archival tissue but can be difficult to work with in traditional NGS assays.
Here we detail workflows using both fresh and fixed patient samples to rapidly produce a diverse set of multiomics results including genomics, epigenomics, transcriptomics and proteomics. For fresh blood draws, this starts with automated sample handling and processing to ensure high viability and yield of PBMCs, along with simultaneous plasma separation and collection. Samples are then aliquoted and simultaneously processed for whole exome sequencing, single cell RNA sequencing, epigenetic characterization and Olink biomarker analysis. For fixed tissues, FFPE blocks were serially sliced into various FFPE slides, with a single slide H&E stained. Individual slides were then utilized for genome, epigenome, single cell RNA-seq, and digital spatial profiling. Genome information was captured using hybrid capture based approaches followed by deep NGS on an Illumina platform and analyzed for a variety of variants and tumor mutational burden. DNA methylation was detailed using target capture probes targeting DNA methylation sites. Single cell approaches were applied to explore the transcriptome using 10X Genomics scRNAseq kit, and Digital Spatial Profiling (DSP) was done using the NanoString GeoMx® Whole Transcriptome Atlas and immunostaining.
With these robust workflows, all these datatypes can be produced within days of fresh or fixed sample receipt using minimal sample amounts. High throughput integrative omics workflows, as described here, drive greater insights in human health, allowing for a rapid combined approach to address the biological questions at hand.
•Classical blood culture testing is still the gold standard in the diagnosis of sepsis•Alternative approaches seem to be more sensitive and faster•The threshold for positive testing through the ...molecular approach seems to be lower
Classical blood culture testing is still the gold standard in correct and timely diagnosis of the responsible microorganisms in sepsis.
In this case (a patient with a colon perforation and severe peritonitis with septic shock), an alternative approach (cell-free DNA next-generation sequencing from full blood samples, NGS) showed the responsible microorganisms, whereas the classical blood culture testing remainedstayed sterile. Interestingly, samples from the abdominal fluid showed the same bacteria as NGS.
These findings may be interpreted as that the threshold for positive testing is lower through the molecular approach than through culture techniques; however, more studies are necessary to prove this theory.
GATA2 deficiency can often predispose individuals to hematologic malignancies. While many pathogenic/likely pathogenic germline variants have been characterized in GATA2 deficiency, less is known ...about other variants that arise during clonal evolution of hematologic malignancies or as potential dual diagnoses in these patients. Here, we present two cases enrolled in the NIAID Centralized Sequencing Program with GATA2 variants and additional pathogenic/likely pathogenic variants in WT1 and KRAS detected via genome sequencing.
The first case is a 9-year-old female with myelodysplastic syndrome with monosomy 7, pulmonary alveolar proteinosis, and sepsis. Genome sequencing in the proband detected 3 rare variants in GATA2: one pathogenic heterozygous NM_032638.5:c.202del (p.Ala68ArgfsTer12) frameshift variant, one heterozygous c.1031_1087dup (p. Arg344_Arg362dup) duplication variant of uncertain significance (VUS), and one apparently somatic mosaic c.1306_1307insTCCA p. (His436LeufsTer101) frameshift VUS. Two variants were also detected in WT1: a likely pathogenic, apparently somatic mosaic, NM_024426.6: c.1159_1163del (p.Ala387Ter) nonsense variant and a heterozygous c.887G>A (p.Ser296Asn) missense VUS. The WT1 p.Ser296Asn variant was previously published in a female affected with Wilms tumor who inherited the variant from her unaffected father, however our participant does not have a personal history of Wilms tumor.
The second case is a 6-year-old male diagnosed with acute myeloid leukemia (AML). Genome sequencing detected a heterozygous GATA2 NM_032638.5(GATA2):c.575C>T (p.Ser192Phe) missense VUS that has not been previously published in individuals with GATA2 deficiency. Additionally, a pathogenic, apparently somatic mosaic, NM_004985.5: c.173C>T (p.Thr58Ile) missense variant in KRAS was detected, which has been reported in the literature as a germline variant in individuals with Noonan syndrome and cardiofaciocutaneous syndrome. The variant allele fraction of the KRAS p.Thr58Ile variant was 27%, however it is unknown if this variant could be somatic mosaic in association with the subject's AML clone, or if the subject has a genetic diagnosis of RAS-associated autoimmune lymphoproliferative syndrome (RALD). Future studies include segregation analysis in family members.
These cases highlight the importance of additional variants found in patients with GATA2 variants that may be contributing to dual diagnoses or acting as secondary mutations via clonal evolution of myeloid malignancies.
The organization of nucleosomes in eukaryotic chromatin is thought to play a critical role in the regulation of the biological function of the chromatin. Because of this potential role in regulation, ...a number of techniques have been developed, which combine chromatin fragmentation around nucleosomes with next-generation sequencing to map the location of nucleosomes in chromatin. In this section, a procedure using a kit from New England Biolabs (NEB NEXT Ultra II FS DNA library prep Kit) to fragment chromatin in preparation for next-generation sequencing is described and compared to other available procedures for mapping nucleosome location.
ROS proto‐oncogene 1, receptor tyrosine kinase (
ROS1
) rearrangements are a crucial therapeutic target in non‐small cell lung cancer (NSCLC). However, there is limited comprehensive analysis of the ...molecular patterns of
ROS1
fusions. This study aimed to address this gap by analysing 135
ROS1
fusions from 134 Chinese NSCLC patients using next‐generation sequencing (NGS). The fusions were categorized into common and uncommon based on their incidence. Our study revealed, for the first time, a unique distribution preference of breakpoints within
ROS1
, with common fusions occurring in introns 31–33 and uncommon fusions occurring in introns 34 and 35. Additionally, we identified previously unknown breakpoints within intron 28 of
ROS1
. Furthermore, we identified a close association between the distribution patterns of fusion partners and breakpoints on
ROS1
, providing important insights into the molecular landscape of
ROS1
fusions. We also confirmed the presence of inconsistent breakpoints in
ROS1
fusions between DNA‐based NGS and RNA‐based NGS through rigorous validation methods. These inconsistencies were attributed to alternative splicing resulting in out‐of‐frame or exonic
ROS1
fusions. These findings significantly contribute to our understanding of the molecular characteristics of
ROS1
fusions, which have implications for panel design and the treatment of NSCLC patients with
ROS1
rearrangements.
Background AML is a complex and heterogeneous disease. The KRAS gene is one of the important genes in the pathogenesis of acute myeloid leukemia (AML). Mutant RAS can promote oncogenesis via ...different mechanisms including oncogenic transcription, cell cycle progression, cellular survival, growth, metabolism, and cell migration. Therefore, it is important to identify the genomic landscape of AML. The aim of the study is to identify KRAS variants in AML and their association with clinic pathological criteria and possible effects on prognosis using NGS.Method Hotspot mutations in the KRAS gene were studied using Ion S5 next-generation sequencing system. Bone marrow samples of newly diagnosed AML patients were collected to identify hotspot mutations in the KRAS gene. DNA amplicons were subjected to sequencing and were analyzed using ion torrent software. Patients were classified according to the FAB classification system. Patients are also classified according to the cytogenetic groups and the ELN risk stratification system.Results KRAS mutations were detected in exon 2, 3, whereas no mutations in KRAS exon 1. Interestingly, Novel mutations were detected in KRAS in AML Egyptian patients. Also, there was no statistically significant association of RAS mutations with different clinical and prognostic parameters. However, KRAS mutant patients tended to have increased PB WBC counts, percentage of PB, and bone marrow blasts.Conclusion NGS is considered a useful tool to identify KRAS variants that could be useful for risk stratification and tailored therapy in AML patients
Breast implant-associated anaplastic large cell lymphoma (BIA-ALCL) is a rare form of T-cell lymphoma that occurs after implantation of breast prostheses. We performed comprehensive next generation ...sequencing based genomic characterization of 11 cases of BIA-ALCL including sequence variant detection on 180 genes frequently mutated in haematological malignancy, genome-wide copy number assessment, structural variant detection involving the T-cell receptor loci and TRB deep-sequencing. We observed sequence variants leading to JAK/STAT activation in 10 out of 11 patients. We also observed germline
mutations in two cases. In addition we detected a recurrent copy number loss involving RPL5 as well as copy number amplifications involving
RANK (in 2 cases),
,
,
and
. In summary, our comprehensive genomic characterisation of 11 cases of BIA-ALCL has provided insight into potential pathobiological mechanisms (JAK/STAT, MYC and TP53) as well as identifying targets for future therapeutic intervention (TNFRSF11A, PDGFRA) in this rare entity.
von Willebrand disease (VWD) is the most frequent inherited bleeding disorder, with an estimated symptomatic prevalence of 1 per 1000 in the general population. VWD is characterized by defects in the ...quantity, quality, or multimeric structure of von Willebrand factor (VWF), a glycoprotein being hemostatically essential in circulation. VWD is classified into 3 principal types: low VWF/type 1 with partial quantitative deficiency of VWF, type 3 with virtual absence of VWF, and type 2 with functional abnormalities of VWF, being classified as 2A, 2B, 2M, and 2N. A new VWD type has been officially recognized by the ISTH SSC on von Willebrand factor which has also been discussed by the joint ASH/ISTH/NHF/WFH 2021 guidelines (ie, type 1C), indicating patients with quantitative deficiency due to an enhanced VWF clearance. With the advent of next-generation sequencing technologies, the process of genetic diagnosis has substantially changed and improved accuracy. Therefore, nowadays, patients with type 3 and severe type 1 VWD can benefit from genetic testing as much as type 2 VWD. Specifically, genetic testing can be used to confirm or differentiate a VWD diagnosis, as well as to provide genetic counseling. The focus of this manuscript is to discuss the current knowledge on VWD molecular pathophysiology and the application of genetic testing for VWD diagnosis.
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