Detection of Low-Copy Human Virus DNA upon Prolonged Formalin Fixation

Formalin fixation, albeit an outstanding method for morphological and molecular preservation, induces DNA damage and cross-linking, which can hinder nucleic acid screening. This is of particular concern in the detection of low-abundance targets, such as persistent DNA viruses. In the present study, we evaluated the analytical sensitivity of viral detection in lung, liver, and kidney specimens from four deceased individuals. The samples were either frozen or incubated in formalin (±paraffin embedding) for up to 10 days. We tested two DNA extraction protocols for the control of efficient yields and viral detections. We used short-amplicon qPCRs (63–159 nucleotides) to detect 11 DNA viruses, as well as hybridization capture of these plus 27 additional ones, followed by deep sequencing. We observed marginally higher ratios of amplifiable DNA and scantly higher viral genoprevalences in the samples extracted with the FFPE dedicated protocol. Based on the findings in the frozen samples, most viruses were detected regardless of the extended fixation times. False-negative calls, particularly by qPCR, correlated with low levels of viral DNA (<250 copies/million cells) and longer PCR amplicons (>150 base pairs). Our data suggest that low-copy viral DNAs can be satisfactorily investigated from FFPE specimens, and encourages further examination of historical materials.

Optimization of capture protocols across species targeting up to 32000 genes and their extension to pooled DNA

In-solution based capture is becoming a method of choice for sequencing targeted sequence. We assessed and optimized a capture protocol in 20 different species from 6 different plant genus using kits from 20,000 to 200,000 baits targeting from 300 to 32,000 genes. We evaluated both the effectiveness of the capture protocol and the fold enrichment in targeted sequences. We proposed a protocol with multiplexing up to 96 samples in a single hybridization and showed it was an efficient and cost-effective strategy. We also extended the use of capture to pools of 100 samples and proved the efficiency of the method to assess allele frequency. Using a set of various organisms with different genome sizes, we demonstrated a correlation between the percentage of on-target reads vs. the relative size of the targeted sequences. Altogether, we proposed methods, strategies, cost-efficient protocols and statistics to better evaluate and more effectively use hybridization capture.

The FORCE Panel: An All-in-One SNP Marker Set for Confirming Investigative Genetic Genealogy Leads and for General Forensic Applications

The FORensic Capture Enrichment (FORCE) panel is an all-in-one SNP panel for forensic applications. This panel of 5422 markers encompasses common, forensically relevant SNPs (identity, ancestry, phenotype, X- and Y-chromosomal SNPs), a novel set of 3931 autosomal SNPs for extended kinship analysis, and no clinically relevant/disease markers. The FORCE panel was developed as a custom hybridization capture assay utilizing ~20,000 baits to target the selected SNPs. Five non-probative, previously identified World War II (WWII) cases were used to assess the kinship panel. Each case included one bone sample and associated family reference DNA samples. Additionally, seven reference quality samples, two 200-year-old bone samples, and four control DNAs were processed for kit performance and concordance assessments. SNP recovery after capture resulted in a mean of ~99% SNPs exceeding 10X coverage for reference and control samples, and 44.4% SNPs for bone samples. The WWII case results showed that the FORCE panel could predict first to fifth degree relationships with strong statistical support (likelihood ratios over 10,000 and posterior probabilities over 99.99%). To conclude, SNPs will be important for further advances in forensic DNA analysis. The FORCE panel shows promising results and demonstrates the utility of a 5000 SNP panel for forensic applications.

The FORCE panel: An all-in-one SNP marker set for confirming investigative genetic genealogy leads and for general forensic applications

The FORensic Capture Enrichment (FORCE) panel is an all-in-one SNP panel for forensic applications. This panel of 5,422 markers encompasses common, forensically relevant SNPs (identity, ancestry, phenotype, X- and Y-chromosomal SNPs), a novel set of 3,931 autosomal SNPs for extended kinship analysis, and no clinically rele-vant/disease markers. The FORCE panel was developed as a custom hybridization capture assay utilizing ~20,000 baits to target the selected SNPs. Five non-probative, previously identified World War II (WWII) cases were used to assess the kinship panel. Each case included one bone sample and associated family reference DNA samples. Additionally, seven reference quality samples, two 200-year-old bone samples, and four control DNAs were processed for kit performance and concordance assessments. SNP recovery after capture resulted in a mean of ~99% SNPs exceeding 10X coverage for reference and control samples, and 44.4% SNPs for bone samples. The WWII case results showed that the FORCE panel could predict 1st to 5th degree relationships with strong statisti-cal support (likelihood ratios over 10,000 and posterior probabilities over 99.99%). To conclude, SNPs will be important for further advances in forensic DNA analysis. The FORCE panel shows promising results and demonstrates the utility of a 5,000 SNP panel for forensic applications.

PATH-37. DISTINCT GENOMIC SUBCLASSES OF HIGH-GRADE/PROGRESSIVE MENINGIOMAS: NF2-ASSOCIATED, NF2-EXCLUSIVE, AND NF2-AGNOSTIC

BACKGROUND Genomic studies of high-grade/progressive meningiomas have reported a heterogeneous mutation spectrum. Here, we present a genomic survey of one of the largest multi-institutional cohorts of high-grade/progressive meningiomas to date. METHODS 850 high-grade/progressive meningiomas, including 441 WHO grade 2 and 176 WHO grade 3 meningiomas and 220 progressive WHO grade 1 meningiomas, were tested as part of a clinical testing program by hybridization capture of 406 cancer-related genes. Information from histopathology review and patient clinical data was assessed. RESULTS Genomic analyses converged to identify at least three distinct patterns of biologically-aggressive meningiomas. The first and most common contained NF2-mutant tumors (n = 426, 50%), was associated with male sex (64.4%, p = 0.0001) and often harbored additional mutations in CDKN2A/B (24%), and the chromatin regulators ARID1A (9%), and KDM6A (6%). A second group (NF2-agnostic) featured TERTp (n = 56) or TP53 mutations (n = 25) and were either NF2-mutant or wild-type, and displayed no association with either sex. The remaining group generally lacked NF2 mutations, and accounted for 40% of the cases-with three subgroups. One consistent primarily of grade 3 lesions harboring alterations in chromatin regulators BAP1 (n = 22) or PBRM1 (n = 16). A second subgroup contained AKT1 (n = 26), PIK3CA (n = 14) and SMO (n = 7) mutant skull-based meningiomas, and a third mixed subgroup included 237 meningiomas with a heterogeneous spectrum of low frequency and non-recurrent alterations. CONCLUSIONS Our findings indicate that the patterns of genomic alterations in high-grade/progressive meningiomas commonly group into three different categories. The most common NF2-associated canonical group frequently harbored CDKN2A/B alterations, which is potentially amenable to targeted therapies. An NF2-agnostic group harbored frequent TERTp and TP53 mutations. The final subclass, distinct from the canonical NF2 mutant associated pathway, was partly characterized by BAP1/PBRM1 alterations (rhabdoid/papillary histology) or skull-base disease.

Recovery of Chloroplast Genomes from Medieval Millet Grains Excavated from the Areni-1 Cave in Southern Armenia

Broomcorn millet (Panicum miliaceum L.) was domesticated in northern China at least 7,000 years ago and was subsequentially adopted as a cereal in many areas throughout Eurasia. One such locale is Areni-1 an archaeological cave site in Southern Armenia, a region that has an important history in crop domestication. The rich botanical material found at Areni-1 includes grains identified by morphology as broomcorn millet that were 14C dated to the medieval era (873 ± 36 CE and 1118 ± 35 CE). To retrace the demographic history of these broomcorn millet samples, we used ancient DNA extraction and hybridization capture enrichment to sequence and assemble three chloroplast genomes from the Areni-1 grains and then compared these sequences to 50 modern chloroplast genomes. Overall, the chloroplast genomes contained a low amount of diversity and little inference on broomcorn demography could be made. However, in a phylogeny the chloroplast genomes separated into two clades with strong bootstrap support, similar to what has been reported for nuclear DNA from broomcorn millet. In a haplotype network, the chloroplast genomes of two accessions of wild (undomesticated) broomcorn millet contained a relatively large number of variants, 11 SNPs. These SNPs were not present in the domesticated varieties, suggesting these wild accessions may not be directly related to the lineages that underwent domestication or that broomcorn millet may have undergone a domestication bottleneck resulting in lost diversity in the chloroplast genome. These results demonstrate that broomcorn millet from archaeological sites can preserve DNA for at least 1000 years and serve as a genetic resource to study the domestication of this cereal crop.

Exploring the Past Biosphere of Chew Bahir/Southern Ethiopia: Cross-Species Hybridization Capture of Ancient Sedimentary DNA from a Deep Drill Core

Eastern Africa has been a prime target for scientific drilling because it is rich in key paleoanthropological sites as well as in paleolakes, containing valuable paleoclimatic information on evolutionary time scales. The Hominin Sites and Paleolakes Drilling Project (HSPDP) explores these paleolakes with the aim of reconstructing environmental conditions around critical episodes of hominin evolution. Identification of biological taxa based on their sedimentary ancient DNA (sedaDNA) traces can contribute to understand past ecological and climatological conditions of the living environment of our ancestors. However, sedaDNA recovery from tropical environments is challenging because high temperatures, UV irradiation, and desiccation result in highly degraded DNA. Consequently, most of the DNA fragments in tropical sediments are too short for PCR amplification. We analyzed sedaDNA in the upper 70 m of the composite sediment core of the HSPDP drill site at Chew Bahir for eukaryotic remnants. We first tested shotgun high throughput sequencing which leads to metagenomes dominated by bacterial DNA of the deep biosphere, while only a small fraction was derived from eukaryotic, and thus probably ancient, DNA. Subsequently, we performed cross-species hybridization capture of sedaDNA to enrich ancient DNA (aDNA) from eukaryotic remnants for paleoenvironmental analysis, using established barcoding genes (cox1 and rbcL for animals and plants, respectively) from 199 species that may have had relatives in the past biosphere at Chew Bahir. Metagenomes yielded after hybridization capture are richer in reads with similarity to cox1 and rbcL in comparison to metagenomes without prior hybridization capture. Taxonomic assignments of the reads from these hybridization capture metagenomes also yielded larger fractions of the eukaryotic domain. For reads assigned to cox1, inferred wet periods were associated with high inferred relative abundances of putative limnic organisms (gastropods, green algae), while inferred dry periods showed increased relative abundances for insects. These findings indicate that cross-species hybridization capture can be an effective approach to enhance the information content of sedaDNA in order to explore biosphere changes associated with past environmental conditions, enabling such analyses even under tropical conditions.

Targeted Hybridization Capture of SARS-CoV-2 and Metagenomics Enables Genetic Variant Discovery and Nasal Microbiome Insights

This is the first FDA emergency-use-authorized hybridization capture-based next-generation sequencing (NGS) assay to detect the SARS-CoV-2 genome. Viral metagenomics and the novel hybrid capture NGS-based assay, along with its research-use-only analysis, can provide important genetic insights into SARS-CoV-2 and other emerging pathogens and improve surveillance and early detection, potentially preventing or mitigating new outbreaks.

Application of Blood Group Genotyping by Next-Generation Sequencing in Various Immunohaematology Cases

<b><i>Background:</i></b> Next-generation sequencing (NGS) technology has been recently introduced into blood group genotyping; however, there are few studies using NGS-based blood group genotyping in real-world clinical settings. In this study, we applied NGS-based blood group genotyping into various immunohaematology cases encountered in routine clinical practice. <b><i>Methods:</i></b> This study included 4 immunohaematology cases: ABO subgroup, ABO chimerism, antibody to a high-frequency antigen (HFA), and anti-CD47 interference. We designed a hybridization capture-based NGS panel targeting 39 blood group-related genes and applied it to the 4 cases. <b><i>Results:</i></b> NGS analysis revealed a novel intronic variant (NM_020469.3:c.29-10T&#x3e;G) in a patient with an A_el phenotype and detected a small fraction of <i>ABO</i>*<i>A1.02</i> (approximately 3–6%) coexisting with the major genotype <i>ABO</i>*<i>B.01</i>/<i>O.01.02</i> in dizygotic twins. In addition, NGS analysis found a homozygous stop-gain variant (NM_004827.3:c.376C&#x3e;T, p.Gln126*; <i>ABCG2</i>*<i>01N.01</i>) in a patient with an antibody to an HFA; consequently, this patient’s phenotype was predicted as Jr(a−). Lastly, blood group phenotypes predicted by NGS were concordant with those determined by serology in 2 patients treated with anti-CD47 drugs. <b><i>Conclusion:</i></b> NGS-based blood group genotyping can be used for identifying <i>ABO</i> subgroup alleles, low levels of blood group chimerism, and antibodies to HFAs. Furthermore, it can be applied to extended blood group antigen matching for patients treated with anti-CD47 drugs.