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PubMed2025

Global biological sample collections from tuberculosis studies: a scoping review.

Progress in tuberculosis vaccine development is hindered by the incomplete understanding of protective immunity and other disease mechanisms. An interconnected network of sample biorepositories from tuberculosis studies could help to address these gaps. To assess the feasibility of such a resource, we conducted a scoping review of tuberculosis observational studies and vaccine clinical trials. The included studies collected at least one biological sample from tuberculosis cases, contacts, or controls and had more than 100 participants. We contacted the corresponding authors of these studies to determine the sample availability and interest in interconnected biorepositories. For the period 2014-24, we identified 104 observational studies and 18 vaccine trials that collected biological samples from 35 075 tuberculosis cases, 39 450 contacts or controls, and 45 628 trial participants across 43 countries. The commonly collected samples were blood, human genomic DNA, RNA, and sputum. Interest among the contacted investigators was high. Interconnected sample biorepositories could facilitate large-scale investigations and accelerate progress towards tuberculosis vaccine development.

Humans↗

PubMed2026

AISP position statement: Standardising biological sample collection and handling for advanced diagnostics and multi-omic analyses in pancreatic cancer.

The quality of biological samples is a major determinant of analytical reliability and translational relevance in patients with pancreatic ductal adenocarcinoma (PDAC). However, variability in specimen procurement, handling, transport, processing, and storage can substantially affect tissue integrity and the robustness of downstream analyses. This paper, promoted by the Pathology and Basic Science Task Force of the Italian Association for the Study of the Pancreas (AISP), brings together experts in pathology, molecular biology, translational research, medical oncology, and gastroenterology to provide practical recommendations for the collection, handling, and pre-analytical management of biological samples. Draft recommendations were discussed during dedicated working group meetings and approved by consensus among all authors, supported by key literature. The document identifies the biological specimen as the critical link between patient care, pathology, and research, and provides guidance for clinicians and professionals involved in sample procurement and processing. By addressing the requirements of different analytical platforms, including genomics, organoid generation, immunophenotyping, pharmacogenomics, and multiplex/spatial analyses, this paper aims to reduce pre-analytical variability, improve diagnostic accuracy, and enhance the clinical and translational value of molecular investigations in pancreatic cancer. Standardised procedures across centres may facilitate comparable data collection, support multicentre studies, and strengthen collaboration between clinicians, pathologists, and research laboratories.

Biobanking↗

PubMed2025

Metaproteomic Analysis to Assess the Impact of Storage Media on Human Gut Microbiome in Fecal Samples.

The human gut microbiome is a diverse community of microorganisms residing in the gastrointestinal tract. The storage condition of fecal samples may impact the taxonomic and protein compositions of microbiomes in these samples. Here, we performed a mass spectrometry-based metaproteomic study to assess the impact of storage media on human gut microbiome in fecal samples. We evaluated FDA-authorized OMNIgene·GUT (OG), phosphate-buffered saline (PBS), and RNALater (RNAL) buffers and identified 38,185 microbial peptides corresponding to 7348 microbial proteins, which matched 16 phyla, 20 classes, 50 orders, 104 families, 332 genera, and 453 species. We found a high similarity among the fecal microbiomes preserved in OG, PBS, and RNAL in terms of the identification of proteins, taxa, and functional annotations. Both alpha and beta diversity suggested the high similarity among samples stored in the three media. Nonetheless, we also found some notable differences among buffers regarding the abundances of a few taxon groups. A partial human proteome (over 400 proteins) was identified in the fecal samples, with most of these proteins associated with the membrane and extracellular regions. The findings indicate the similarity among microbiomes in the fecal samples stored in OG, PBS, and RNAL regarding proteome profile, taxa, and functional capacity. SUMMARY: This study thoroughly analyzed and compared the metaproteomes of fecal samples preserved at -80°C in PBS, RNALater, and OMNIgene·GUT Dx buffers, offering novel insights into the effectiveness of these buffers in maintaining the stability and composition of the human gut microbiome. We found a high similarity in the identification and quantification of proteins, taxa, and functional annotations across the three buffers, with notable quantitative differences highlighting subtle yet important variations in preservation efficacy. The unique datasets and findings could offer valuable revelations into the impact of fecal sample preservation on translational and clinical analyses of the human gut microbiome.

Humans↗

PubMed2026

Determinants of nucleic acid quality in formalin-fixed paraffin-embedded bladder urothelial carcinoma specimens: effects of specimen type, fixation conditions, and storage time.

Formalin-fixed paraffin-embedded (FFPE) tissue is the principal substrate for cancer genomic profiling, yet pre-analytical factors affect nucleic acid quality. In our workflow, transurethral resection (TUR) specimens of bladder urothelial carcinoma are fixed promptly but exposed to electrocautery, whereas robot-assisted radical cystectomy specimens are refrigerated overnight before fixation; we compared nucleic acid quality between these specimen types. We analyzed 56 FFPE primary bladder urothelial carcinoma specimens (33 TUR, 23 cystectomy). DNA quality was assessed by the DNA Integrity Number (DIN) and short-to-long cycle threshold ratio (S/L Ct ratio), and RNA quality by the RNA Integrity Number (RIN) and percentage of RNA fragments ≥200 nucleotides (DV200), with group comparisons, Spearman correlations, and multivariable regression (specimen type, fixation duration, storage time). The median DIN (4.4 vs. 3.8, p = 0.004) and S/L Ct ratio (0.943 vs. 0.894, p < 0.001) were higher in TUR than cystectomy specimens; the RIN was also higher (1.9 vs. 1.7, p = 0.040), though low in both groups, and DV200 did not differ (50.3% vs. 47.7%, p = 0.934). In multivariable analysis, TUR specimen type independently predicted higher DIN (β = +0.460, p = 0.040) and S/L Ct ratio (β = +0.442, p = 0.009), whereas longer storage time independently decreased the S/L Ct ratio (β = -0.513, p < 0.001) and RIN (β = -0.352, p = 0.010). In our institutional workflow, TUR specimens showed higher DNA quality metrics than robot-assisted radical cystectomy specimens subjected to overnight refrigerated pre-fixation delay, whereas this workflow-associated advantage did not clearly extend to DV200-defined RNA fragment-length quality. Storage time was associated with selected aspects of nucleic acid deterioration.

Humans↗

PubMed2026

A rare and atypical case of long-distance indirect DNA transfer: Contamination from an investigator never present at the scene.

To maximize the usefulness of DNA obtained from biological samples in forensic genetics, it is crucial to avoid DNA contamination throughout all procedures, from sample collection at crime scenes to STR profile generation in DNA laboratories. This study reports a rare and atypical case of DNA contamination in a forensic setting. During the analysis of biological evidence from a cold case preserved for 18 years, the STR profile obtained from the surface of a plastic bag matched that of an investigator, identified through the DNA elimination database. Case reconstruction confirmed that the investigator-who was located 80 km from the DNA laboratory and had never entered the crime scene or the sample storage room-was not a suspect and that the obtained STR profile originated from contamination. The most plausible explanation for the contamination was indirect transfer: investigator's DNA had adhered to a colleague's clothing and was subsequently dislodged and deposited onto the surface of the plastic bag as the colleague approached the sample pretreatment area. This study integrates trace DNA profiling of challenged samples with rapid contamination investigation and proposes prevention and control measures. This case underscores that, although DNA is widely regarded as the "gold standard" in forensic genetics, its interpretation must be considered within the context of the entire case. Conclusions should not be drawn based solely on a single DNA result.

Humans↗

PubMed2026

Saliva versus lesion swabs for PCR diagnosis of acute-phase clade Ib mpox in Uganda: a prospective matched hospital cohort study.

BACKGROUND: As clade Ib mpox expands through HIV-affected populations in east and central Africa, diagnostic specimen selection should balance accuracy, accessibility, and operational feasibility in outbreak settings. Here, we aimed to compare the diagnostic performance of matched plasma, saliva, genital, anal, and skin specimens during the acute rash phase of clade Ib mpox to identify clinically practical and high-yield sampling approaches for outbreak response and clinical care. METHODS: We conducted a prospective cohort study of 155 adults (median age 30 years, IQR 25-36) hospitalised at Uganda's national mpox referral hospital. The specimens were collected between March 3 and April 10, 2025, during the clade Ib outbreak. All participants were admitted with suspected mpox and were subsequently confirmed by MPXV PCR. We collected 836 clinical specimens (acid citrate dextrose plasma, saliva, genital swabs, anal swabs, and skin swabs) during the acute phase (visit 1; 14 days [SD 2] after systemic symptom onset) and at approximately 3 months (visit 2). A matched acute-phase subset (n=80) provided concurrent plasma, saliva, genital, and skin specimens for within-participant comparisons. MPXV DNA was quantified by F3L real-time quantitative PCR, and cycle threshold (Ct) values were compared using paired Wilcoxon signed-rank tests. Whole-genome sequencing of selected acute specimens confirmed clade assignment. FINDINGS: In the matched subset at visit 1, PCR positivity was high in saliva (78 [98%] of 80), skin swabs (78 [98%] of 80), and genital swabs (77 [96%] of 80). Results for the saliva closely mirrored genital and skin swab results, supporting saliva as a high-yield alternative when lesion sampling is painful, operationally difficult, or unacceptable. Plasma had substantially lower sensitivity (34 [43%] of 80) and showed poor agreement with mucocutaneous compartments. At 3 months, persistent MPXV DNA was rare (ten [9%] of 109) and clustered among people with HIV, including the only two participants with persistent plasma positivity. All sequenced genomes clustered within clade Ib. INTERPRETATION: During the established rash phase (14 days [SD 2] after onset), saliva provides diagnostic yield similar to that provided by lesion swabs for clade Ib mpox in this hospitalised cohort. These findings are restricted to this sampling window; further studies are needed to define performance in prodromal, pre-rash, and asymptomatic infection. FUNDING: CEPI through its Centralised Laboratory Network.

Adult↗

PubMed2025

Evaluation of swabbing methods for culture and non-culture-based recovery of multidrug-resistant organisms from environmental surfaces.

OBJECTIVES: Sponge-Sticks (SS) and ESwabs are frequently utilized for detection of multidrug-resistant organisms (MDROs) in the environment. Head-to-head comparisons of SS and ESwabs across recovery endpoints are limited. DESIGN: We compared MDRO culture and non-culture-based recovery from (1) ESwabs, (2) cellulose-containing SS (CS), and (3) polyurethane-containing SS (PCS). METHODS: Known quantities of each MDRO were pipetted on a stainless-steel surface and swabbed by each method. Samples were processed, cultured, and underwent colony counting. DNA was extracted from sample eluates, quantified, and underwent metagenomic next-generation sequencing (mNGS). MDROs underwent whole genome sequencing (WGS). MDRO recovery from paired patient perirectal and PCS-collected environmental samples from clinical studies was determined. SETTING: Laboratory experiment, tertiary medical center, and long-term acute care facility. RESULTS: Culture-based recovery varied across MDRO taxa, it was highest for vancomycin-resistant Enterococcus and lowest for carbapenem-resistant Pseudomonas aeruginosa (CRPA). Culture-based recovery was significantly higher for SS compared to ESwabs except for CRPA, where all methods performed poorly. Nucleic acid recovery varied across methods and MDRO taxa. Integrated WGS and mNGS analysis resulted in successful detection of antimicrobial resistance genes, construction of high-quality metagenome-assembled genomes, and detection of MDRO genomes in environmental metagenomes across methods. In paired patient and environmental samples, multidrug-resistant Pseudomonas aeruginosa (MDRP) environmental recovery was notably poor (0/123), despite detection of MDRP in patient samples (20/123). CONCLUSIONS: Our findings support the use of SS for the recovery of MDROs. Pitfalls of each method should be noted. Method selection should be driven by MDRO target and desired endpoint.

Humans↗

PubMed2025

Comparative performance of sponge versus flocked swabs for culture-based and metagenomic detection of microbial contamination in the healthcare environment.

BACKGROUND: Identifying optimal methods for sampling surfaces in the healthcare environment is critical for future research requiring the identification of multidrug-resistant organisms (MDROs) on surfaces. METHODS: We compared 2 swabbing methods, use of a flocked swab versus a sponge-stick, for recovery of MDROs by both culture and recovery of bacterial DNA via quantitative 16S polymerase chain reaction (PCR). This comparison was conducted by assessing swab performance in a longitudinal survey of MDRO contamination in hospital rooms. Additionally, a laboratory-prepared surface was also used to compare the recovery of each swab type with a matching surface area. RESULTS: Sponge-sticks were superior to flocked swabs for culture-based recovery of MDROs, with a sensitivity of 80% compared to 58%. Similarly, sponge-sticks demonstrated greater recovery of Staphylococcus aureus from laboratory-prepared surfaces, although the performance of flocked swabs improved when premoistened. In contrast, recovery of bacterial DNA via quantitative 16S PCR was greater with flocked swabs by an average of 3 log copies per specimen. CONCLUSIONS: The optimal swabbing method of environmental surfaces differs by method of analysis. Sponge-sticks were superior to flocked swabs for culture-based detection of bacteria but inferior for recovery of bacterial DNA.

Humans↗

PubMed2026

Minimizing decompression and warming during deep seawater collection increases abundance and activity of autochthonous bacteria and archaea.

The deep ocean hosts autochthonous pressure-adapted microorganisms that are unique to this environment, as well as allochthonous pressure-sensitive members transported from shallow depths by vertical advection and particle-sinking. However, conventional sampling instruments decompress and warm deep-sea samples during retrieval, potentially altering microbial properties when studied ex situ. Here, we assess this potential sampling bias by comparing seawater microbial communities collected with or without measures aimed at minimizing pressure and temperature effects. When compared to samples collected under pressurized conditions, conventional sampling (using Niskin bottles) was found to affect prokaryotic cells retrieved by reducing their total numbers, diminishing protein synthesis activity (>10%), and also causing overall shifts in the community composition. The most significant compositional change was a >20% decrease in metagenomic archaeal representation (TACK-group/Thaumarchaeota/Nitrososphaerota). Deep-sea bacterial groups had mixed responses to preserving pressure during retrieval, with some groups exhibiting higher representation when samples were maintained pressurized (e.g. members of the family Pelagibacteraceae, unclassified Thiotricales, Thioglobaceae, and Chitinophagaceae), whereas others increased their representation when decompressed (e.g. Burkholderiaceae, Comamonadaceae, and Oxalobacteraceae). This study reveals the existence of bias introduced by the complete decompression of samples retrieved with traditional instrumentation, as well as a decrease in overall bacterial activity when samples are completely decompressed during retrieval. Additionally, incubations lasting for >24 h were shown to transform the original prokaryotic community composition. Precautions addressing these effects are necessary to enhance the reliability of ex situ measurements and improve our understanding of deep-sea microbial ecology and biogeochemistry.

Seawater↗

PubMed2026

Bycatch in a bottle: what taxa are recoverable from metabarcoding DNA in historical invertebrate collection preservative fluid?

Natural history museum collections are invaluable repositories of biodiversity, offering insights into life on Earth. Genomic approaches provide powerful tools to characterize biodiversity in these collections. However using these collections for genomics without damaging specimens is a challenge. Here, we develop and test non-destructive DNA metabarcoding methods to capture biodiversity from the preservative fluids of archived insect collections ('Bycatch'). We optimized workflows for extracting and amplifying the partial CO1 locus (CO1) and fungal ITS1 locus from ethanol-based preservative fluids, validating ethanol preparation methods, comparing DNA extraction kits, and refining PCR protocols. Our results demonstrate that from museum collections with low DNA yields, CO1 and fungal ITS1 loci can often be recovered from preservative fluids, and we present detailed methodology and workflows. We test metabarcoding success to recover taxa in several museum collections ranging in age and storage condition. This is to support the State of California's effort to catalog and sequence all insects and fungi, building baselines of California biodiversity with help from museum collections. Lastly, we investigate the complementarity of metabarcoding water versus ethanol and morphological identifications aimed to capture benthic macroinvertebrate biodiversity in streams. Our findings highlight that DNA metabarcoding of the preservative fluid is a non-destructive tool for capturing biodiversity in historical specimens, but there are limitations on the overlaps between DNA results and physical contents, where morphological identification still reigns in taxon counts, but metabarcoding sometimes provides more taxonomic resolution, and can be used to track DNA from other organisms such as fungi beyond the directly surveyed specimens.

Animals↗

PubMed2026

Evaluation of bone preparation approaches using length-based analysis and targeted sequencing for forensic human identification of historic skeletal remains.

Advances in DNA technology have significantly enhanced the forensic community's ability to develop genetic profiles from unidentified human skeletal remains. However, sampling requires mechanical grinding of hard tissues before DNA isolation. This processing can compromise genetic profiles, particularly in aged bones. We compared the industry-standard pulverization method with an alternative powder-free preparation involving prolonged demineralization and subsequent slicing of 19th-century cortical bone. Data from DNA quantification, STR genotyping, and targeted SNP sequencing were used to evaluate powdered samples versus demineralized slices from paired human bones. Average human DNA yields for pulverized samples and demineralized slices were 0.032 ng and 0.692 ng, respectively. Demineralized slices recovered more amplifiable DNA than traditional homogenization methods (p < 0.05). No pulverized samples produced STR profiles, whereas demineralized slices from the same bone samples yielded partial profiles. Samples underwent DNA repair, library preparation, and hybridization capture using the FORensic Capture Enrichment (FORCE) panel. Applying low-coverage (1X) analysis of high-throughput sequencing (HTS) data, demineralized slices outperformed those prepared by traditional pulverization methods (p < 0.05) and substantially increased the information recovered compared with conventional STR analysis methods. Based on HTS data from pulverized samples, DNA fragment length ranged from 27 to 95 bp, and FORCE SNP recovery was 33.23%. In contrast, for demineralized slices, DNA fragment length ranged from 85 to 114 bp, and FORCE SNP recovery was 83.24%. The required reagents and equipment are typically available in forensic labs, and the workflow outlined herein significantly increases the success of DNA recovery from challenging skeletal samples.

Humans↗

PubMed2026

Ambient temperature storage of individual parasitic nematode larvae for whole genome sequencing.

Soil-transmitted helminth (STH) infections are a major public health burden, and there are programmes of mass drug administration that attempt to ameliorate the harm that they cause. There has been increasing use of genomics to study STH infections and other parasitic nematodes, with particular interest in whole genome sequencing (WGS). For such studies, samples are commonly stored frozen, but in settings where these infections are endemic this can be difficult, and so there would be advantages to having ambient temperature storage methods. We investigated two ambient temperature storage methods - FTA cards and DESS buffer - for infective larvae of the rat parasites Nippostrongylus brasiliensis and Strongyloides ratti, prior to DNA extraction and then WGS. Our results showed that for individual larvae stored on FTA cards or in DESS buffer, this resulted in a lower proportion of sequence reads that mapped to the reference genomes, compared to the frozen control samples. Generally, for individual larvae, DESS-storage resulted in better sequencing results than FTA-storage. However, for pools of 10 or 50 larvae, then these ambient temperature storage methods generally resulted in comparable sequence read mapping to the frozen control samples.

Animals↗

PubMed2024

Effectiveness of stabilization methods for the immediate and short-term preservation of bovine fecal and upper respiratory tract genomic DNA.

Previous research on stabilization methods for microbiome investigations has largely focused on human fecal samples. There are a few studies using feces from other species, but no published studies investigating preservation of samples collected from cattle. Given that microbial taxa are differentially impacted during storage it is warranted to study impacts of preservation methods on microbial communities found in samples outside of human fecal samples. Here we tested methods of preserving bovine fecal respiratory specimens for up to 2 weeks at four temperatures (room temperature, 4°C, -20°C, and -80°C) by comparing microbial diversity and community composition to samples extracted immediately after collection. Importantly, fecal specimens preserved and analyzed were technical replicates, providing a look at the effects of preservation method in the absence of biological variation. We found that preservation with the OMNIgene®•GUT kit resulted in community structure most like that of fresh samples extracted immediately, even when stored at room temperature (~20°C). Samples that were flash-frozen without added preservation solution were the next most representative of original communities, while samples preserved with ethanol were the least representative. These results contradict previous reports that ethanol is effective in preserving fecal communities and suggest for studies investigating cattle either flash-freezing of samples without preservative or preservation with OMNIgene®•GUT will yield more representative microbial communities.

Cattle↗

PubMed2026

URMD-Seq: A high-throughput method for scalable detection of ultra-rare mutations in the human mitochondrial genome.

The study of mitochondrial genetics has long been limited to polymorphisms and high frequency mutations owing in part to technical and technological limitations in reliably detecting and quantifying rare somatic mutations. Over the past decade or so, the study of rare somatic mitochondrial DNA (mtDNA) variants has expanded and continues to garner increasing interest in a wide range of research fields. Here, we describe Ultra-Rare Mutation Detection-Sequencing (URMD-Seq), a high-throughput method that combines unique molecular identifier (UMI)-based library preparation and Next Generation Sequencing (NGS) for the accurate and scalable detection of ultra-rare mutations in the mtDNA control region. Our method exploits degenerate primers to label individual mtDNA molecules. This is followed by several purification, quantification and amplification steps, to obtain high quality amplicons for sequencing on the Illumina MiSeq platform. Our approach enables the use of total genomic DNA extract as starting point for the assay, overcoming the need for organelle isolation and/or mtDNA enrichment, hence broadening the type of specimen that can be studied, while offering cost and time benefits. The assay described herein has been demonstrated to reliably measure variants present at on average 0.09%, but as low as 0.03%, variant allele frequency in a variety of tissues, including fresh and frozen biobanked specimens. Using this protocol, library preparation of 300 specimens can be completed by a single individual with general nucleic acid handling experience in approximately 20 days. Given its flexibility and scalability, URMD-Seq is particularly well suited for epidemiological studies using a large number of specimens.

Humans↗

PubMed2026

Exploratory single-nucleus multiomics analysis of myeloid cell states associated with neoadjuvant chemotherapy response in pancreatic ductal adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) continues to be one of the most lethal human malignancies, with the vast majority of patients ineligible for immunotherapy. Tumour-associated macrophages (TAMs) are key regulators of the PDAC tumour microenvironment (TME), yet their transcriptional and epigenomic heterogeneity in the context of chemotherapy response is poorly understood. Therefore, we performed an exploratory single nucleus multiomics analysis of PDAC tumors stratified by histopathologic response to neoadjuvant chemotherapy. METHODS: Surgical resection specimens from PDAC patients were classified as responders or non-responders using the American College of Pathologists (CAP) histopathologic criteria. Frozen tissue underwent simultaneous snRNA-seq and snATAC-seq on the 10x Genomics Chromium Single Cell Multiome platform, followed by downstream analyses such as differential gene expression, GO and hallmark pathway enrichment, pseudotime trajectory inference and ChromVAR transcription factor motif analysis. RESULTS: Multiomics profiling of 30 840 high-quality nuclei revealed a myeloid compartment that differed in composition and transcriptional state between CAP-defined responders and non-responders in this small cohort. We observed a trend toward higher LAM-like state proportions in the responders than non-responders (38.4% vs. 26.7%), although this disparity did not achieve statistical significance. The transcriptional programs of the responder myeloid cells are associated with phagocytosis and lipid handling. Chromatin accessibility analysis further suggested candidate response-associated transcription factor motif accessibility patterns. CONCLUSIONS: Neoadjuvant-treated PDAC tumours from CAP-defined responders in this cohort myeloid landscape with apparent enrichment of LAM-like states and immune-activating transcriptional/epigenetic programs. However, these findings are preliminary and hypothesis-generating because of the small cohort size, heterogeneous treatment regimens, absence of matched pre-treatment biopsies, and lack of knockout validation. Larger treatment cohorts and functional/mechanistic studies are needed to determine whether LAM-like myeloid programs contribute to chemotherapy response or reflect a consequence of chemotherapy treatment.

Humans↗