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.
Highlights • Impressive advances in NGS have enabled an immense diversity of novel applications. • The barrier of the $1000 genome has recently been broken. • Important novel tools for clinical ...diagnostics based on NGS are appearing. • Third-generation technologies may further revolutionize genomics research. • Significant challenges for NGS remain, in particular data storage and processing.
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.
•The present study is the first report of the complete mitochondrial genome of B. kachuga (16,517 bp) construed via next-generation sequencing (NGS) approach from eggshell DNA.•Phylogenetic ...relationships among major lineages of Testudines were reconstructed using 32 other complete mitochondrial genomes.•Maximum likelihood phylogenetic tree strongly supports each family in different clades and also revealed a close relationship between the Pangashura and Batagur genus.
The Batagur kachuga (B. kachuga), commonly known as the Red-crowned roofed turtle, is a critically endangered species native to India and its neighboring countries like Bangladesh, and Nepal. The present study is the first report of the complete mitochondrial genome of B. kachuga (16,517 bp) construed via the next-generation sequencing (NGS) approach from eggshell DNA. There are 22 transfer RNAs (tRNAs), 2 ribosomal RNAs (rRNAs), 13 protein-coding genes (PCGs), and one putative control region (CR/D-loop) in the mitogenome. The CR region from the current study reveals conserved TAS, CD, and CSB domains and two AT-rich tandem repeat regions. Most genes are encoded in the heavy strand except the NADH dehydrogenase subunit 6 (ND6) gene and seven tRNA genes. Most PCGs start with the initiation codon ATG, except the COI (Cytochrome Oxidase Subunit-I) gene, which starts with the GTG codon. The present investigation also predicts the distinctive cloverleaf structures of tRNAs except for tRNA-Ser1 and tRNA-Ser-2, which lack a DHU arm. The comparative analysis of Ka/Ks with other 33 species from Order Testudines, in relation to B. kachuga, revealed negative selection in most PCGs, indicating a process of preservation and purification that aids in eliminating undesirable or detrimental substitutes. Phylogenetic analysis of this species has been analysed using the complete mitogenome of 33 turtle species. The maximum likelihood phylogenetic tree strongly supports each family in different clades and also reveals a close relationship between the Pangashura and Batagur genera. Our study suggests the generation of genome-wide molecular data, in terms of mitogenomes, SNPs, and SSRs, is needed to improve the understanding of this species and their phylogenetics and evolutionary relationships, which will help to improve the conservation efforts of this species.
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.
Seafood fraud involves mislabeling and false claims. To avoid this deceptive practice, authentication techniques, including DNA-based methods, have been developed. These techniques detect fraud, ...ensure accurate labeling, and maintain product integrity, aiding in species identification, verifying origins, and promoting industry transparency.
This review aims to provide a detailed list of approaches for the authenticity of mackerel species due to the prevalence of mislabeling and substitution. Utilizing techniques like DNA, we can identify Scomber species by analyzing specific genetic markers. This work advances the evaluation of existing molecular markers through in silico simulations, while also purposing novel primers designed for a more accurate authentication of processed mackerel products.
With advances in techniques, we are seeing significant advances in food authentication methods. DNA barcoding has seen substantial evolution, thanks to improvements in databases and NGS sequencing technologies. The advent of DNA metabarcoding is also revolutionizing species identification in highly processed seafoods. Using more comprehensive primers in Metabarcoding can enhance the assessment of scombrid products. In recent times, SNP-based approaches with microfluidic enrichment, DNA-based biosensors and microbiome sequencing techniques have emerged as key tools for assessing the authenticity and identifying the origins of seafood products.
•Global mackerel seafood labelling discrepancies affect consumer transparency•Molecular techniques refine Scomber species identification•NGS advancements enhance seafood mackerel authentication for fraud detection•DNA barcoding is crucial for fraud detection and ensures mackerel authenticity•In silico evaluation: DNA metabarcoding for Scomber species
