DNA Enrichment Research Articles

Application of fetal cell-free DNA enrichment in non-invasive prenatal screening: experience from a single center in Eastern China.

Application of fetal cell-free DNA enrichment in non-invasive prenatal screening: experience from a single center in Eastern China.

Strain-Level Analysis of Bifidobacterium spp. from Gut Microbiomes of Adults with Differing Lactase Persistence Genotypes.

One of the strongest associations between human genetics and the gut microbiome is a greater relative abundance of Bifidobacterium in adults with lactase gene (LCT) single nucleotide polymorphisms (SNPs) associated with lactase nonpersistence (GG genotypes), versus lactase persistence (AA/AG genotypes). To gain a finer-grained phylogenetic resolution of this association, we interrogated 1,680 16S rRNA libraries and 245 metagenomes from gut microbiomes of adults with various lactase persistence genotypes. We further employed a novel genome-capture-based enrichment of Bifidobacterium DNA from a subset of these metagenomes, including monozygotic (MZ) twin pairs, each sampled 2 or 3 times. B. adolescentis and B. longum were the most abundant Bifidobacterium species regardless of host LCT genotype. LCT genotypes could not be discriminated based on relative abundances of Bifidobacterium species or Bifidobacterium community structure. Three distinct metagenomic analysis methods of Bifidobacterium-enriched DNA revealed intraindividual temporal stability of B. longum, B. adolescentis, and B. bifidum strains against the background of a changeable microbiome. Two of our three methods also observed greater strain sharing within MZ twin pairs than within unrelated individuals for B. adolescentis, while no method revealed an effect of host LCT genotype on Bifidobacterium strain composition. Our results support a "rising tide lifts all boats" model for the dominant bifidobacteria in the adult gut: their higher abundance in lactase-nonpersistent than in lactase-persistent individuals results from an expansion at the genus level. Bifidobacterium species are known to be transmitted from mother to child and stable within individuals in infancy and childhood: our results extend this stability into adulthood.IMPORTANCE When humans domesticated animals, some adapted genetically to digest milk into adulthood (lactase persistence). The gut microbiomes of people with lactase-persistent genotypes (AA or AG) differ from those with lactase-nonpersistent genotypes (GG) by containing fewer bacteria belonging to the bifidobacteria, a group which contains beneficial species. Here, we asked if the gut microbiomes of adults with GG and AA/AG genotypes differ in the species of bifidobacteria present. In particular, we used a novel technique which allowed us to compare bifidobacteria in adults at the strain level, without the traditional need for culturing. Our results show that the GG genotype enhances the abundance of bifidobacteria regardless of species. We also noted that a person's specific strains are recoverable several years later, and twins can share the same ones. Given that bifidobacteria are inherited from mother to child, strain stability over time in adulthood suggests long-term, multigenerational inheritance.

DNA Thermo-Protection Facilitates Whole-Genome Sequencing of Mycobacteria Direct from Clinical Samples.

Mycobacterium tuberculosis is the leading cause of death from bacterial infection. Improved rapid diagnosis and antimicrobial resistance determination, such as by whole-genome sequencing, are required. Our aim was to develop a simple, low-cost method of preparing DNA for sequencing direct from M. tuberculosis-positive clinical samples (without culture). Simultaneous sputum liquefaction, bacteria heat inactivation (99°C/30 min), and enrichment for mycobacteria DNA were achieved using an equal volume of thermo-protection buffer (4 M KCl, 0.05 M HEPES buffer, pH 7.5, 0.1% dithiothreitol [DTT]). The buffer emulated intracellular conditions found in hyperthermophiles, thus protecting DNA from rapid thermodegradation, which renders it a poor template for sequencing. Initial validation experiments employed mycobacteria DNA, either extracted or intracellular. Next, mock clinical samples (infection-negative human sputum spiked with 0 to 105Mycobacterium bovis BCG cells/ml) underwent liquefaction in thermo-protection buffer and heat inactivation. DNA was extracted and sequenced. Human DNA degraded faster than mycobacteria DNA, resulting in target enrichment. Four replicate experiments achieved M. tuberculosis detection at 101 BCG cells/ml, with 31 to 59 M. tuberculosis complex reads. Maximal genome coverage (>97% at 5× depth) occurred at 104 BCG cells/ml; >91% coverage (1× depth) occurred at 103 BCG cells/ml. Final validation employed M. tuberculosis-positive clinical samples (n = 20), revealing that initial sample volumes of ≥1 ml typically yielded higher mean depths of M. tuberculosis genome coverage, with an overall range of 0.55 to 81.02. A mean depth of 3 gave >96% 1-fold tuberculosis (TB) genome coverage (in 15/20 clinical samples). A mean depth of 15 achieved >99% 5-fold genome coverage (in 9/20 clinical samples). In summary, direct-from-sample sequencing of M. tuberculosis genomes was facilitated by a low-cost thermo-protection buffer.

Natural attenuation of lead by microbial manganese oxides in a karst aquifer

Lead is a toxic environmental contaminant associated with current and historic mine sites. Here we studied the natural attenuation of Pb in a limestone cave system that receives drainage from the ancient Priddy Mineries, UK. Extensive deposits of manganese oxides were observed to be forming on the cave walls and as coatings in the stream beds. Analysis of these deposits identified them as birnessite (δ-MnO2), with some extremely high concentrations of sorbed Pb (up to 56 wt%) also present. We hypothesised that these cave crusts were actively being formed by microbial Mn(II)-oxidation, and to investigate this the microbial communities were characterised by DNA sequencing, enrichment and isolation experiments. The birnessite deposits contained abundant and diverse prokaryotes and fungi, with ~5% of prokaryotes and ~ 10% of fungi closely related to known heterotrophic Mn(II)-oxidisers. A substantial proportion (up to 17%) of prokaryote sequences were assigned to groups known as autotrophic ammonia and nitrite oxidisers, suggesting that nitrogen cycling may play an important role in contributing energy and carbon to the cave crust microbial communities and consequently the formation of Mn(IV) oxides and Pb attenuation. Enrichment and isolation experiments showed that the birnessite deposits contained Mn(II)-oxidising microorganisms, and two isolates (Streptomyces sp. and Phyllobacterium sp.) could oxidise Mn(II) in the presence of 0.1 mM Pb. Supplying the enrichment cultures with acetate as a source of energy and carbon stimulated Mn(II)-oxidation, but excess organics in the form of glucose generated aqueous Mn(II), likely via microbial Mn(IV)-reduction. In this karst cave, microbial Mn(II)-oxidation contributes to the active sequestration and natural attenuation of Pb from contaminated waters, and therefore may be considered a natural analogue for the design of wastewater remediation systems and for understanding the geochemical controls on karst groundwater quality, a resource relied upon by billions of people across the globe.

MEIRLOP: improving score-based motif enrichment by incorporating sequence bias covariates

BackgroundMotif enrichment analysis (MEA) identifies over-represented transcription factor binding (TF) motifs in the DNA sequence of regulatory regions, enabling researchers to infer which transcription factors can regulate transcriptional response to a stimulus, or identify sequence features found near a target protein in a ChIP-seq experiment. Score-based MEA determines motifs enriched in regions exhibiting extreme differences in regulatory activity, but existing methods do not control for biases in GC content or dinucleotide composition. This lack of control for sequence bias, such as those often found in CpG islands, can obscure the enrichment of biologically relevant motifs.ResultsWe developed Motif Enrichment In Ranked Lists of Peaks (MEIRLOP), a novel MEA method that determines enrichment of TF binding motifs in a list of scored regulatory regions, while controlling for sequence bias. In this study, we compare MEIRLOP against other MEA methods in identifying binding motifs found enriched in differentially active regulatory regions after interferon-beta stimulus, finding that using logistic regression and covariates improves the ability to call enrichment of ISGF3 binding motifs from differential acetylation ChIP-seq data compared to other methods. Our method achieves similar or better performance compared to other methods when quantifying the enrichment of TF binding motifs from ENCODE TF ChIP-seq datasets. We also demonstrate how MEIRLOP is broadly applicable to the analysis of numerous types of NGS assays and experimental designs.ConclusionsOur results demonstrate the importance of controlling for sequence bias when accurately identifying enriched DNA sequence motifs using score-based MEA. MEIRLOP is available for download from https://github.com/npdeloss/meirlop under the MIT license.

Genome Wide Analysis of the Transcriptional Profiles in Different Regions of the Developing Rice Grains

BackgroundRice is an important food source for humans worldwide. Because of its nutritional and agricultural significance, a number of studies addressed various aspects of rice grain development and grain filling. Nevertheless, the molecular processes underlying grain filling and development, and in particular the contributions of different grain tissues to these processes, are not understood.Main TextUsing RNA-sequencing, we profiled gene expression activity in grain tissues comprised of cross cells (CC), the nucellar epidermis (NE), ovular vascular trace (OVT), endosperm (EN) and the aleurone layer (AL). These tissues were dissected using laser capture microdissection (LCM) at three distinct grain development stages. The mRNA expression datasets offer comprehensive and new insights into the gene expression patterns in different rice grain tissues and their contributions to grain development. Comparative analysis of the different tissues revealed their similar and/or unique functions, as well as the spatio-temporal regulation of common and tissue-specific genes. The expression patterns of genes encoding hormones and transporters indicate an important role of the OVT tissue in metabolite transport during grain development. Gene co-expression network prediction on OVT-specific genes identified several distinct and common development-specific transcription factors. Further analysis of enriched DNA sequence motifs proximal to OVT-specific genes revealed known and novel DNA sequence motifs relevant to rice grain development.ConclusionTogether, the dataset of gene expression in rice grain tissues is a novel and useful resource for further work to dissect the molecular and metabolic processes during rice grain development.

Improvement of the Detection Technique of <i>Listeria monocytogenes</i> through Modification of the Enrichment Medium and DNA Extraction Buffer

Improvement of the Detection Technique of <i>Listeria monocytogenes</i> through Modification of the Enrichment Medium and DNA Extraction Buffer

An enhanced target-enrichment bait set for Hexacorallia provides phylogenomic resolution of the staghorn corals (Acroporidae) and close relatives

Targeted enrichment of genomic DNA can profoundly increase the phylogenetic resolution of clades and inform taxonomy. Here, we redesign a custom bait set previously developed for the cnidarian class Anthozoa to more efficiently target and capture ultraconserved elements (UCEs) and exonic loci within the subclass Hexacorallia. We test this enhanced bait set (targeting 2476 loci) on 99 specimens of scleractinian corals spanning both the “complex” (Acroporidae, Agariciidae) and “robust” (Fungiidae) clades. Focused sampling in the staghorn corals (genus Acropora) highlights the ability of sequence capture to inform the taxonomy of a clade previously deficient in molecular resolution. A mean of 1850 (±298) loci were captured per taxon (955 UCEs, 894 exons), and a 75% complete concatenated alignment of 96 samples included 1792 loci (991 UCE, 801 exons) and ~1.87 million base pairs. Maximum likelihood and Bayesian analyses recovered robust molecular relationships and revealed that species-level relationships within the Acropora are incongruent with traditional morphological groupings. Both UCE and exon datasets delineated six well-supported clades within Acropora. The enhanced bait set will facilitate investigations of the evolutionary history of many important groups of reef corals, particularly where previous molecular marker development has been unsuccessful.

Evaluation of un-methylated DNA enrichment in sequencing of African swine fever virus complete genome

African swine fever is a febrile hemorrhagic fever disease that is caused by the African swine fever virus (ASFV) and is lethal for domestic pigs and wild boar. ASFV also infects soft ticks of the genus Ornithodoros, some species of which can act as a vector for ASFV. Whole genome sequencing of ASFV is a challenge because, due to the size difference of the host genome versus the viral genome, the higher proportion of host versus virus DNA fragments renders the virus sequencing poorly efficient. A novel approach of DNA enrichment, based on the separation of methylated and un-methylated DNA, has been reported but without an evaluation of its efficacy. In this study, the efficiency of the un-methylated DNA enrichment protocol was evaluated for pig and tick samples infected by ASFV. As expected, fewer reads corresponding to ASFV were found in the methylated fraction compared to the un-methylated fraction. However, the sequencing coverage of the un-methylated fraction was not improved compared to the untreated DNA. In our hands, the ASFV DNA enrichment was inefficient for tick samples and very limited for pig samples. This enrichment process represents extra work and cost without a significant improvement of ASFV genome coverage. The efficiency of this enrichment approach and the cost/benefit ratio are discussed.

The Absence of C-5 DNA Methylation in Leishmania donovani Allows DNA Enrichment from Complex Samples.

Cytosine C5 methylation is an important epigenetic control mechanism in a wide array of eukaryotic organisms and generally carried out by proteins of the C-5 DNA methyltransferase family (DNMTs). In several protozoans, the status of this mechanism remains elusive, such as in Leishmania, the causative agent of the disease leishmaniasis in humans and a wide array of vertebrate animals. In this work, we showed that the Leishmania donovani genome contains a C-5 DNA methyltransferase (DNMT) from the DNMT6 subfamily, whose function is still unclear, and verified its expression at the RNA level. We created viable overexpressor and knock-out lines of this enzyme and characterized their genome-wide methylation patterns using whole-genome bisulfite sequencing, together with promastigote and amastigote control lines. Interestingly, despite the DNMT6 presence, we found that methylation levels were equal to or lower than 0.0003% at CpG sites, 0.0005% at CHG sites, and 0.0126% at CHH sites at the genomic scale. As none of the methylated sites were retained after manual verification, we conclude that there is no evidence for DNA methylation in this species. We demonstrated that this difference in DNA methylation between the parasite (no detectable DNA methylation) and the vertebrate host (DNA methylation) allowed enrichment of parasite vs. host DNA using methyl-CpG-binding domain columns, readily available in commercial kits. As such, we depleted methylated DNA from mixes of Leishmania promastigote and amastigote DNA with human DNA, resulting in average Leishmania:human enrichments from 62× up to 263×. These results open a promising avenue for unmethylated DNA enrichment as a pre-enrichment step before sequencing Leishmania clinical samples.

Abstract 969: Pharmacologic induction of innate immune signaling via STING directly drives homologous recombination deficiency

Abstract Poly (ADP-ribose) polymerase inhibitors (PARPi) are FDA approved in a subset of patients with ovarian cancer or metastatic breast cancers who harbor BRCA gene mutations. Although these mutations, which generate homologous recombination deficiencies (HRD), have been the main predictor to PARPi sensitivity, responses to therapy have not been durable and have failed for the majority of sporadic triple negative breast cancers (TNBC). We previously reported that DNA methyltransferase inhibitor (DNMTi) azacytidine (Aza) improves the efficacy of a new generation of PARPi, Talazoparib (Tal), through increased trapping of cytotoxic PARP-DNA complexes in both BRCA-mutant and -proficient TNBC. These trapped complexes lead to increased and persistent levels of lethal double strand breaks (DSBs), suggesting that DSB repair may also be impaired with this treatment. Indeed, Aza/Tal treatment in BRCA-proficient TNBC cell lines significantly downregulates expression of HR and Fanconi Anemia (FA) genes, notably FANCD2, and decreases HR activity, thus generating HRD. DNMTi have also been established to induce a viral mimicry response which upregulate type I interferon (IFN) signaling and production of inflammatory cytokines. In the present study, we elucidate the link between Aza/Tal facilitated HRD and induction of innate immune and inflammatory related genes, mediated through a STING dependent mechanism, which we term as a pathogen mimicry response (PMR). Gene set enrichment analysis of RNA-Seq data derived from mono- and combination-treated TNBC cell line MDA-MB-231, reveals enrichment of innate immune and cytosolic DNA sensing pathways with significant increases of TNFα/NF-κB and IFNαβ gene sets. Overlap between HRD and immune related signaling was evaluated using the STRING database, which reveals a significant interaction specifically between FA and TNFα/NF-κB and IFNαβ pathway genes. Tal driven cytosolic DNA as well as an Aza augmentation in STING protein expression, emerges as the key node in Aza/Tal induced innate immune signaling. Transient knockdown of STING is able to significantly rescue Aza- and combination-induced repression of FANCD2. Critically, in the presence of FANCD2 rescue facilitated by STING-specific inhibition, Aza-induced HRD was also reversed. These STING perturbation data suggest that Aza-induced HRD is dependent on presence of competent STING signaling. This inverse relationship was validated in both METABRIC TNBC dataset and TCGA data sets including lung adenocarcinoma and lung squamous cell carcinoma, colon adenocarcinoma, and AML, thus suggesting broad applicability of this observed transcriptional program independent of pharmacologic intervention. Induction of PMR to drive HRD suggests that DNMTi-PARPi therapy strategies can expand the therapeutic scope of PARPi to encompass treatment of BRCA-proficient cancers. Citation Format: Lena J. McLaughlin, Aksinija A. Kogan, Stephen B. Baylin, Michael J. Topper, Feyruz V. Rassool. Pharmacologic induction of innate immune signaling via STING directly drives homologous recombination deficiency [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 969.

Evaluation of beta-thalassemia in the fetus through cffDNA with multiple polymorphisms as a haplotype in the beta-globin gene.

Invasive biopsy during the pregnancy is associated with an abortion risk of approximately 1% for the fetus. Free fetal DNA in maternal plasma is an excellent source of genetic material for prenatal molecular diagnoses. This study was conducted to investigate beta-thalassemia mutation in the fetus through maternal blood with multiple polymorphisms as haplotypes in the beta-globin gene. In this study, a total of 33 beta-thalassemia carrier (minor) couples were genotyped by ARMS-PCR for IVSII-IG>A mutation. During pregnancy, 10mL of blood was collected from pregnant women, and DNA was extracted by the magnetic bead-based extraction, and fetal DNA was enriched with AMPure XP kit. Five polymorphisms in 4 haplotype groups were evaluated by the Sanger Sequencing method. Finally, results were compared with those of the invasion method. Participants in study were 33 couples, mean age of the men was 26±5 years, and mean age of women was 23±4 years, and mean MCV, MCH, HbA2 blood parameters were 62.4±5.3, 19.6±3.1, 4.2±2.1 respectively. A total of 33 fetuses were genotyped for IVSII-IG>A mutation. Nine fetuses were affected, 10 fetuses were normal and 14 fetuses were carrier of beta-thalassemia. Sensitivity and specificity of Sanger Sequencing were equal to 88.8% and 91.6% respectively. Positive and negative predictive values were obtained as 80% and 95.6%, respectively. Mutational status of the fetus can be assessed by determining inheritance of paternally-derived alleles based on detection of haplotype-associated SNP in maternal plasma. Magnetic-based DNA extraction and fetal DNA enrichment are very simple and easy to perform and have satisfactory accuracy.

Selective enrichment of zein gene of maize from cereal products using magnetic support having pyrrolidinyl peptide nucleic acid probe

Here, we describe DNA enrichment of the zein gene from maize using pyrrolidinyl peptide nucleic acid (PNA) immobilized on a magnetic solid support as a capture element. Magnetite nanoparticles (MNP) with a capacity of 373 pmolPNA/mg and coated with poly(N-acryloylglycine) (PNAG) showed a good response to magnetic field. The PNA probe immobilized on the MNP discriminated between non-complementary and complementary DNA using fluorophore-tagged DNA as a model. We applied this system for the enrichment of the zein gene from maize in eight cereal product samples. After DNA desorption from the MNP, and its amplification via polymerase chain reaction (PCR), gel electrophoresis indicated that only cereal samples containing the zein gene from maize yielded positive results, indicating a high binding specificity between the PNA used and the complementary DNA. This PNA-functionalized MNP is potentially useful as an effective nano-solid support for DNA enrichment from other samples.

A Practical Method to Implement Strain-Level Metagenomics-Based Foodborne Outbreak Investigation and Source Tracking in Routine.

The management of a foodborne outbreak depends on the rapid and accurate identification of the responsible food source. Conventional methods based on isolation of the pathogen from the food matrix and target-specific real-time polymerase chain reactions (qPCRs) are used in routine. In recent years, the use of whole genome sequencing (WGS) of bacterial isolates has proven its value to collect relevant information for strain characterization as well as tracing the origin of the contamination by linking the food isolate with the patient’s isolate with high resolution. However, the isolation of a bacterial pathogen from food matrices is often time-consuming and not always successful. Therefore, we aimed to improve outbreak investigation by developing a method that can be implemented in reference laboratories to characterize the pathogen in the food vehicle without its prior isolation and link it back to human cases. We tested and validated a shotgun metagenomics approach by spiking food pathogens in specific food matrices using the Shiga toxin-producing Escherichia coli (STEC) as a case study. Different DNA extraction kits and enrichment procedures were investigated to obtain the most practical workflow. We demonstrated the feasibility of shotgun metagenomics to obtain the same information as in ISO/TS 13136:2012 and WGS of the isolate in parallel by inferring the genome of the contaminant and characterizing it in a shorter timeframe. This was achieved in food samples containing different E. coli strains, including a combination of different STEC strains. For the first time, we also managed to link individual strains from a food product to isolates from human cases, demonstrating the power of shotgun metagenomics for rapid outbreak investigation and source tracking.

Prediction-based highly sensitive CRISPR off-target validation using target-specific DNA enrichment

CRISPR effectors, which comprise a CRISPR-Cas protein and a guide (g)RNA derived from the bacterial immune system, are widely used for target-specific genome editing. When the gRNA recognizes genomic loci with sequences that are similar to the target, deleterious mutations can occur. Off-target mutations with a frequency below 0.5% remain mostly undetected by current genome-wide off-target detection techniques. Here we report a method to effectively detect extremely small amounts of mutated DNA based on predicted off-target-specific amplification. In this study, we used various genome editors to induce intracellular genome mutations, and the CRISPR amplification method detected off-target mutations at a significantly higher rate (1.6~984 fold increase) than an existing targeted amplicon sequencing method. In the near future, CRISPR amplification in combination with genome-wide off-target detection methods will allow detection of genome editor-induced off-target mutations with high sensitivity and in a non-biased manner.

Shedding light on dark genes: enhanced targeted resequencing by optimizing the combination of enrichment technology and DNA fragment length

The exome contains many obscure regions difficult to explore with current short-read sequencing methods. Repetitious genomic regions prevent the unique alignment of reads, which is essential for the identification of clinically-relevant genetic variants. Long-read technologies attempt to resolve multiple-mapping regions, but they still produce many sequencing errors. Thus, a new approach is required to enlighten the obscure regions of the genome and rescue variants that would be otherwise neglected. This work aims to improve the alignment of multiple-mapping reads through the extension of the standard DNA fragment size. As Illumina can sequence fragments up to 550 bp, we tested different DNA fragment lengths using four major commercial WES platforms and found that longer DNA fragments achieved a higher genotypability. This metric, which indicates base calling calculated by combining depth of coverage with the confidence of read alignment, increased from hundreds to thousands of genes, including several associated with clinical phenotypes. While depth of coverage has been considered crucial for the assessment of WES performance, we demonstrated that genotypability has a greater impact in revealing obscure regions, with ~1% increase in variant calling in respect to shorter DNA fragments. Results confirmed that this approach enlightened many regions previously not explored.

4058 Enhanced efficiency of large-scale clinical proteomic studies: when less is more

OBJECTIVES/GOALS: Large-scale clinical proteomic studies of cancer tissues often entail complex workflows and are resource-intensive. In this study we analyzed ovarian tumors using an emerging, high-throughput proteomic technology termed SWATH. We compared SWATH with the more widely used iTRAQ workflow based on robustness, complexity, ability to detect differential protein expression, and the elucidated biological information. METHODS/STUDY POPULATION: Proteomic measurements of 103 clinically-annotated high-grade serous ovarian cancer (HGSOC) tumors previously genomically characterized by The Cancer Genome Atlas were conducted using two orthogonal mass spectrometry-based proteomic methods: iTRAQ and SWATH. The analytical differences between the two methods were compared with respect to relative protein abundances. To assess the ability to classify the tumors into subtypes based on proteomic signatures, an unbiased molecular taxonomy of HGSOC was established using protein abundance data. The 1,599 proteins quantified in both datasets were classified based on z-score-transformed protein abundances, and the emergent protein modules were characterized using weighted gene-correlation network analysis and Reactome pathway enrichment. RESULTS/ANTICIPATED RESULTS: Despite the greater than two-fold difference in the analytical depth of each proteomic method, common differentially expressed proteins in enriched pathways associated with the HGSOC Mesenchymal subtype were identified by both methods. The stability of tumor subtype classification was sensitive to the number of analyzed samples, and the statistically stable subgroups were identified by the data from both methods. Additionally, the homologous recombination deficiency-associated enriched DNA repair and chromosome organization pathways were conserved in both data sets. DISCUSSION/SIGNIFICANCE OF IMPACT: SWATH is a robust proteomic method that can be used to elucidate cancer biology. The lower number of proteins detected by SWATH compared to iTRAQ is mitigated by its streamlined workflow, increased sample throughput, and reduced sample requirement. SWATH therefore presents novel opportunities to enhance the efficiency of clinical proteomic studies.

Histone post-translational modifications in Silene latifolia X and Y chromosomes suggest a mammal-like dosage compensation system

Silene latifolia is a model organism to study evolutionary young heteromorphic sex chromosome evolution in plants. Previous research indicates a Y-allele gene degeneration and a dosage compensation system already operating. Here, we propose an epigenetic approach based on analysis of several histone post-translational modifications (PTMs) to find the first epigenetic hints of the X:Y sex chromosome system regulation in S. latifolia. Through chromatin immunoprecipitation we interrogated six genes from X and Y alleles. Several histone PTMS linked to DNA methylation and transcriptional repression (H3K27me3, H3K23me, H3K9me2 and H3K9me3) and to transcriptional activation (H3K4me3 and H4K5, 8, 12, 16ac) were used. DNA enrichment (Immunoprecipitated DNA/input DNA) was analyzed and showed three main results: (i) promoters of the Y allele are associated with heterochromatin marks, (ii) promoters of the X allele in males are associated with activation of transcription marks and finally, (iii) promoters of X alleles in females are associated with active and repressive marks. Our finding indicates a transcription activation of X allele and transcription repression of Y allele in males. In females we found a possible differential regulation (up X1, down X2) of each female X allele. These results agree with the mammal-like epigenetic dosage compensation regulation.

Correlation of tumor mutational burden (TMB) with molecular profiling and clinical characteristics in patients with bladder cancer.

e17025 Background: Bladder cancers (BC) are frequently highly mutated. Next generation sequencing (NGS) can both shed light on mutational burden and the specific alterations that provide insights into the underlying biology of individual tumors. Methods: We retrospectively reviewed BC cases assessed with UCSF500, an institutional NGS assay that uses hybrid capture enrichment of target DNA to interrogate approximately 500 frequently mutated cancer genes. Hypermutated tumors were defined as having TMB > 10 mutations/Mb. Fisher’s exact test was used to compare patients (pts) with hypermutated (HM) and non-hypermutated (NHM) tumors. Results: From 2015 to 2019, 74 pts with BC underwent UCSF500 testing; 48 pts were evaluable for TMB, of which 19 pts (40%) had HM tumors. 17/19 pts were evaluable for mutational signatures; all 17 had APOBEC signatures. Signatures were not assessed in NHM tumors due to low TMB. Clinicopathologic characteristics and most common alterations in the two groups are listed in the table. More HM pts had responses to immunotherapy (IO) treatment (86% vs 40%, p = 0.13). Conclusions: In this single-institution BC cohort, HM tumors were common and APOBEC mutational signature was the common underlying biology in HM tumors. There were relevant differences in common alterations between HM and NHM tumors, including more FGFR3 mutations in NHM tumors. HM status and APOBEC signature were suggested as relevant predictive biomarkers of response to IO, which should be investigated further in larger BC cohorts. [Table: see text]

SUN-710 Custom Panel to Diagnosis Genetic Endocrine Disorders in a Tertiary Academic Hospital

Next-generation sequencing (NGS) has been transforming the endocrine diagnostic methodology allowing the genetic testing to assume an exploratory role rather than only a confirmatory one. This is possible due to lower costs and increased yield of information. A way to further increase efficiency and sensitivity for variant detection is the use of a sequencing custom panel selecting specific genes for screening. In endocrine disorders, the complex and intricate genotype-phenotype relations and occurrence of diverse comorbidities made the diagnosis challenging. Our aim is to analyze the efficiency of a multigenic panel for molecular diagnosis of endocrine disorders in patients assisted in a tertiary academic hospital, as well as to train academic and medical faculties in the use of molecular tools. Genomic DNA from 282 patients was extracted from blood sample using standard procedures. Sanger method was previously used to screen some candidate genes in half of the patients. The custom panel was designed with 651 genes using the SureDesign tool (Agilent technologies), either associated with the phenotype (OMIM) or candidate genes that englobes developmental (DD), metabolic (MD), and adrenal (AD) disorders. Libraries were prepared with SureSelectXT Target Enrichment kit (Agilent Technologies). The enriched DNA libraries were sequenced in NextSeq 500 (Illumina) with High Output V2 kit (2 x 150 bp). The raw data was aligned to hg19 with BWA-MEM, variant calling was performed using FreeBayes and annotated with ANNOVAR. Filtering took into consideration the rarity (≤1%) of variants in population databases and those in exonic or splice site regions. Variants found were then classified according ACMG/AMP criteria. The categories of Pathogenic (P) and Likely Pathogenic (LP) were considered for molecular diagnosis, while variants of uncertain significance (VUS) were only reported. The average result of 3 runs was: 159Kmm2 of cluster density, 76.5 % of Q30 and 76.6 Gb of data were generated. The mean coverage depth of the targeted regions in panel sequencing data was 237x (SD±110x), with at least 96.3% of the sequenced bases being covered more than 20-fold. Out of the 282 patients, we identified 65 LP/P variants (23%), 22 VUS (8%) and 195 remained undiagnosed (69%). Considering the solved cases, 54 (19.1%) have DD, 6 (2.1%) have MD and 5 (1.8%) have AD. Taking into account that half of the patients had already been previously screened, the data enable new findings in known genes. The application of a multigenic panel aids the training of medical faculty in an academic hospital by showing the big picture of the molecular pathways behind each disorder. This may be particularly helpful considering the higher diagnosis of DD cases. A precise genetic etiology provides improvement in understanding the disease, guides decisions about prevention or treatment, and brings comfort to the affected families.