Sequencing Libraries Research Articles

#3031 Cell free DNA methylation analysis reveals cerebral cell death during hemodialysis

Abstract Background and Aims Patients treated with hemodialysis demonstrate a significant cognitive decline, likely explained by cerebrovascular disease and hypoperfusion. Despite the significant morbidity and mortality associated, no method exists to detect subclinical cerebral injury. This prevents early diagnosis and precludes mitigation strategies. Following cell death, cell free DNA (cfDNA) is released into the peripheral blood. Since DNA methylation is cell-type specific, cfDNA methylation analysis can reveal the cellular origin of cfDNA and cell-type specific changes in cell death. Here, we evaluate if subclinical cerebral ischemia during hemodialysis is detectable through cfDNA methylome analyses. Method Chronic hemodialysis patients with a high cardiovascular risk profile were studied during a single hemodialysis session. Brain tissue oxygenation was measured continuously by near-infrared spectroscopy using two sensors on the forehead. Plasma samples were taken immediately before and after hemodialysis. cfDNA was extracted from the blood plasma and converted into whole genome bisulfite sequencing libraries using tailored protocols. Next, in-solution target enrichment was applied to analyze 4,989 genomic loci, selected for showing specific methylation in one of 15 tissues or cell types. After sequencing, reads were aligned to the hg38 reference genome using BisMark, and deconvoluted using EMeth-Laplace to disclose the relative contribution of the 15 selected tissues or cell types. Results 41 hemodialysis patients participated in the study, with a mean age of 75 ± 8 years (46% male). The median ultrafiltration volume was 2.3 L (IQR 1.6-3.0 L). After targeted bisulfite sequencing and QC-based filtering, 35 patients remained for cfDNA methylation analysis. In 30 patients, cfDNA concentrations were increased following dialysis. Deconvolution analysis revealed a relative increase in granulocyte-derived cfDNA from 36.7% to 55% before versus after dialysis. Consequently, for most other tissues and cell types we observed a relative decrease in their cfDNA contribution. Interestingly, a subset of patients (n = 13) showed an increase in neuron-derived cfDNA from 0% to 0.26%, while no neuron-derived cfDNA was detectable in the remaining 22 patients. Near-infrared spectroscopy showed a significantly reduced cerebral oxygenation in these 13 patients (P < 0.0001) at 2 separate time windows during hemodialysis treatment (at 20-60 and 207-251 minutes into the session) (Fig.). Conclusion In patients with a high cardiovascular risk profile, cfDNA in peripheral blood increased after hemodialysis. cfDNA methylome analyses suggested this to be due to an increased contribution of granulocytes, indicating elevated levels of NETosis. In addition, we observed more neuron-derived cfDNA after dialysis in about 1 in 3 patients. This was most apparent in patients with a reduced brain tissue oxygenation during hemodialysis. Together, these findings suggest that hypoperfusion during hemodialysis can evoke neuronal cell death, detectable through methylome analyses of cfDNA isolated from a minimally invasively obtainable bodily fluid. Follow-up studies are ongoing to assess if this translates to progressive cognitive impairment.

Expanding the synthetic biology repertoire of a fast-growing cyanobacterium Synechococcus elongatus PCC 11801.

Synechococcus elongatus PCC 11801 is a fast-growing cyanobacterium, exhibiting high tolerance to environmental stresses. We have earlier characterized its genome and analysed its transcriptome and proteome. However, to deploy it as a potential cell factory, it is necessary to expand its synthetic biology toolbox, including promoter elements and ribosome binding sites (RBSs). Here, based on the global transcriptome analysis, 48 native promoters of the genes with high transcript count were characterized using a fluorescent reporter system. The promoters PcpcB, PpsbA1, and P11770 exhibited consistently high fluorescence under all the cultivation conditions. Similarly, from the genome data and proteome analysis, 534 operons were identified. Fifteen intergenic regions exhibiting higher protein expression from the downstream gene were systematically characterized for identifying RBSs, using an operon construct comprising fluorescent protein genes eyfp and mTurq under PcpcB (PcpcB:eyfp:RBS:mTurq:TrrnB). Overall, the work presents promoter and RBS sequence libraries, with varying strengths, to expedite bioengineering of PCC 11801.

Spatial transcriptomics reveals gene interactions and signaling pathway dynamics in rat embryos with anorectal malformation

Anorectal malformation (ARM) is a prevalent early pregnancy digestive tract anomaly. The intricate anatomy of the embryonic cloaca region makes it challenging for traditional high-throughput sequencing methods to capture location-specific information. Spatial transcriptomics was used to sequence libraries of frozen sections from embryonic rats at gestational days (GD) 14 to 16, covering both normal and ARM cases. Bioinformatics analyses and predictions were performed using methods such as WGCNA, GSEA, and PROGENy. Immunofluorescence staining was used to verify gene expression levels. Gene expression data was obtained with anatomical annotations of clusters, focusing on the cloaca region's location-specific traits. WGCNA revealed gene modules linked to normal and ARM cloacal anatomy development, with cooperation between modules on GD14 and GD15. Differential gene expression profiles and functional enrichment were presented. Notably, protein levels of Pcsk9, Hmgb2, and Sod1 were found to be downregulated in the GD15 ARM hindgut. The PROGENy algorithm predicted the activity and interplay of common signaling pathways in embryonic sections, highlighting their synergistic and complementary effects. A competing endogenous RNA (ceRNA) regulatory network was constructed from whole transcriptome data. Spatial transcriptomics provided location-specific cloaca region gene expression. Diverse bioinformatics analyses deepened our understanding of ARM's molecular interactions, guiding future research and providing insights into gene regulation in ARM development.

Dual RNA-seq in filarial nematodes and Wolbachia endosymbionts using RNase H based ribosomal RNA depletion.

Lymphatic filariasis is caused by parasitic nematodes and is a leading cause of disability worldwide. Many filarial worms contain the bacterium Wolbachia as an obligate endosymbiont. RNA sequencing is a common technique used to study their molecular relationships and to identify potential drug targets against the nematode and bacteria. Ribosomal RNA (rRNA) is the most abundant RNA species, accounting for 80-90% of the RNA in a sample. To reduce sequencing costs, it is necessary to remove ribosomal reads through poly-A enrichment or ribosomal depletion. Bacterial RNA does not contain a poly-A tail, making it difficult to sequence both the nematode and Wolbachia from the same library preparation using standard poly-A selection. Ribosomal depletion can utilize species-specific oligonucleotide probes to remove rRNA through pull-down or degradation methods. While species-specific probes are commercially available for many commonly studied model organisms, there are currently limited depletion options for filarial parasites. Here, we performed total RNA sequencing from Brugia malayi containing the Wolbachia symbiont (wBm) and designed ssDNA depletion probes against their rRNA sequences. We compared the total RNA library to poly-A enriched, Terminator 5'-Phosphate-Dependent Exonuclease treated, NEBNext Human/Bacteria rRNA depleted and our custom nematode probe depleted libraries. The custom nematode depletion library had the lowest percentage of ribosomal reads across all methods, with a 300-fold decrease in rRNA when compared to the total RNA library. The nematode depletion libraries also contained the highest percentage of Wolbachia mRNA reads, resulting in a 16-1,000-fold increase in bacterial reads compared to the other enrichment and depletion methods. Finally, we found that the Brugia malayi depletion probes can remove rRNA from the filarial worm Dirofilaria immitis and the majority of rRNA from the more distantly related free living nematode Caenorhabditis elegans. These custom filarial probes will allow for future dual RNA-seq experiments between nematodes and their bacterial symbionts from a single sequencing library.

A Comparative Analysis of Secreted miRNAs Reveals Candidate Biomarkers for Pleural Mesothelioma Detection

Abstract Background MicroRNAs secreted by tumour cells through exosomes or in non-encapsulated form, were shown to promote tumour growth and treatment resistance, underscoring their potential as cancer biomarkers. Aims Here, we compare miRNAs secreted by pleural mesothelioma (PM) primary cells with miRNAs secreted by non-PM cells, in order to identify diagnostic biomarkers of this devastating disease. Methods We established primary cell cultures from pleural effusion of 12 PM and 7 non-PM patients. Secreted miRNAs were profiled as: (1) total secreted miRNA in cell culture supernatant (Sup), and (2) exosomal (Exo) miRNAs. Exosomes were extracted using iZON qEV columns. RNA was extracted with mirVana PARIS kit. Sequencing libraries were prepared using the QIAseq miRNA Library Kit (Qiagen). Reads were mapped on the mature miRNA sequences (MirBaseDB), followed by DESeq2 differential expression analysis. MiRNA target genes were selected using MirDB and functionally annotated using DAVID. Results We identified 309 and 84 deregulated miRNAs in PM-Exo and PM-Sup samples, respectively (p-value <0.05, Fig. 1A). Among the up-regulated miRNAs, we found let-7c-3p and miR-16-5p, which are known tumour suppressors, and miR-23a-3p and miR-30a-5p (Fig. 1B). Interestingly, miR-30a-5p belongs to the same family as miR-30e-5p, which together with miR-23a-3p was previously identified as part of the long survival signature in PM. Additionally, we identified 11 miRNAs that showed up-regulation in both PM-Exo and PM-Sup. Further analysis of these candidate miRNAs revealed that exosomal expression of miR-30a-5p was a significant predictor of patient survival (Fig. 1C). Finally, we identified potential target genes of the 11 up-regulated miRNAs - the target genes were significantly associated with transcriptional regulation and cell division (p-value <0.05). Conclusion Our comparative analysis of secreted miRNAs identified 11 candidate biomarkers for PM. Our findings emphasise the importance of the PM secretome in advancing our understanding of mesothelioma biology and discovering novel cell-free biomarkers. Further in-depth analyses are currently underway.

UCE-derived mitochondrial phylogeny reveals pervasive mito-nuclear discordances in serotine bats (genus Eptesicus) and complex evolutionary history in Eptesicus (Histiotus)

Studies of evolution and biodiversity require solid understanding of species systematics revealed by molecular phylogeny using multilocus genomic data. Multilocus analyses, nevertheless, remain difficult in non-model taxa due to limited access to samples and molecular resources. To help overcome this limitation, ultra-conserved elements (UCEs) have been developed to generate large nuclear datasets and build more robust species phylogenies. Recently, MitoFinder pipeline was developed to further extract mitochondrial genes from the off-target sequences in UCE libraries to allow mito-nuclear comparison and increase the mitochondrial genomic database. Here we applied MitoFinder to published UCE datasets of serotine bats (genus Eptesicus) and focused on E. (Histiotus) whose evolutionary history is poorly understood. Our results showed extensive mito-nuclear discordances in the divergence of major clades in Eptesicus and within E. (Histiotus), indicating potential incomplete lineage sorting and historical mitochondrial introgression within and across subgenera. Moreover, we collected several new samples of E. (Histiotus), including the first molecular data of the recently described E. (H) diaphanopterus, and combined available published sequences to generate the most taxa-complete mitochondrial phylogeny of E. (Histiotus) bats. Results supported the early divergence of E. (H.) magellanicus and the species status of E. (H.) diaphanopterus. In addition, we found strong evidence of cryptic diversity, with potentially new taxa in Peru, Uruguay, and Brazil, which needs to be evaluated in future studies using complementary data. Our study enriched the sequence database of serotine bats and shed light on the hidden diversity and complex evolutionary history of E. (Histiotus).

Readsynth: short-read simulation for consideration of composition-biases in reduced metagenome sequencing approaches

BackgroundThe application of reduced metagenomic sequencing approaches holds promise as a middle ground between targeted amplicon sequencing and whole metagenome sequencing approaches but has not been widely adopted as a technique. A major barrier to adoption is the lack of read simulation software built to handle characteristic features of these novel approaches. Reduced metagenomic sequencing (RMS) produces unique patterns of fragmentation per genome that are sensitive to restriction enzyme choice, and the non-uniform size selection of these fragments may introduce novel challenges to taxonomic assignment as well as relative abundance estimates.ResultsThrough the development and application of simulation software, readsynth, we compare simulated metagenomic sequencing libraries with existing RMS data to assess the influence of multiple library preparation and sequencing steps on downstream analytical results. Based on read depth per position, readsynth achieved 0.79 Pearson’s correlation and 0.94 Spearman’s correlation to these benchmarks. Application of a novel estimation approach, fixed length taxonomic ratios, improved quantification accuracy of simulated human gut microbial communities when compared to estimates of mean or median coverage.ConclusionsWe investigate the possible strengths and weaknesses of applying the RMS technique to profiling microbial communities via simulations with readsynth. The choice of restriction enzymes and size selection steps in library prep are non-trivial decisions that bias downstream profiling and quantification. The simulations investigated in this study illustrate the possible limits of preparing metagenomic libraries with a reduced representation sequencing approach, but also allow for the development of strategies for producing and handling the sequence data produced by this promising application.

SWIGH-SCORE: A translational light-weight approach in computational detection of rearranged immunoglobulin heavy chain to be used in monoclonal lymphoproliferative disorders

We present a lightweight tool for clonotyping and measurable residual disease (MRD) assessment in monoclonal lymphoproliferative disorders. It is a translational method that enables computational detection of rearranged immunoglobulin heavy chain gene sequences.•The swigh-score clonotyping tool emphasizes parallelization and applicability across sequencing platforms.•The algorithm is based on an adaptation of the Smith-Waterman algorithm for local alignment of reads generated by 2nd and 3rd generation of sequencers.For method validation, we demonstrate the targeted sequences of immunoglobulin heavy chain genes from diagnostic bone marrow using serial dilutions of CD138+ plasma cells from a patient with multiple myeloma. Sequencing libraries from diagnostic samples were prepared for the three sequencing platforms, Ion S5 (Thermo Fisher Scientific), MiSeq (Illumina), and MinION (Oxford Nanopore), using the LymphoTrack assay. Basic quality filtering was performed, and a Smith-Waterman-based swigh-score algorithm was developed in shell and C for clonotyping and MRD assessment using FASTQ data files. Performance is demonstrated across the three different sequencing platforms.

Development, validation and application of single molecule molecular inversion probe based novel integrated genetic screening method for 29 common lysosomal storage disorders in India

BackgroundCurrent clinical diagnosis pathway for lysosomal storage disorders (LSDs) involves sequential biochemical enzymatic tests followed by DNA sequencing, which is iterative, has low diagnostic yield and is costly due to overlapping clinical presentations. Here, we describe a novel low-cost and high-throughput sequencing assay using single-molecule molecular inversion probes (smMIPs) to screen for causative single nucleotide variants (SNVs) and copy number variants (CNVs) in genes associated with 29 common LSDs in India.Results903 smMIPs were designed to target exon and exon–intron boundaries of targeted genes (n = 23; 53.7 kb of the human genome) and were equimolarly pooled to create a sequencing library. After extensive validation in a cohort of 50 patients, we screened 300 patients with either biochemical diagnosis (n = 187) or clinical suspicion (n = 113) of LSDs. A diagnostic yield of 83.4% was observed in patients with prior biochemical diagnosis of LSD. Furthermore, diagnostic yield of 73.9% (n = 54/73) was observed in patients with high clinical suspicion of LSD in contrast with 2.4% (n = 1/40) in patients with low clinical suspicion of LSD. In addition to detecting SNVs, the assay could detect single and multi-exon copy number variants with high confidence. Critically, Niemann-Pick disease type C and neuronal ceroid lipofuscinosis-6 diseases for which biochemical testing is unavailable, could be diagnosed using our assay. Lastly, we observed a non-inferior performance of the assay in DNA extracted from dried blood spots in comparison with whole blood.ConclusionWe developed a flexible and scalable assay to reliably detect genetic causes of 29 common LSDs in India. The assay consolidates the detection of multiple variant types in multiple sample types while having improved diagnostic yield at same or lower cost compared to current clinical paradigm.

Genetic diversity and population structure of four Anatolian sheep revealed by genome-wide ddRADseq data

This is the first wide-scale study to reveal next-generation sequencing-based and comprehensive study to reveal genetic diversity, population structure, and phylogenetic relationships of some of Türkiye's indigenous fat-tailed sheep breeds. A total of 80 animals belonging to Akkaraman (AKR), Güney Karaman (GKR), Morkaraman (MKR), and Karakaş (KRK) sheep breeds were genotyped with genome-wide of 296.097 single nucleotide polymorphisms (SNPs) data recovered from double digest restriction site-associated DNA sequencing (ddRADseq) libraries processed by Illumina HiSeq X Ten instrument. Minor allele frequency (MAF) ranged from 0.311 (MKR) to 0.316 (AKR and GKR). Observed heterozygosity (HO) values were higher than expected values (HE) across all populations with a mean of 0.307. Negative inbreeding coefficient (FIS) values were observed in four sheep populations varying between −0.034 (AKR) to −0.060 (MKR). Cross-entropy criterion-based ADMIXTURE analysis demonstrated that four Anatolian sheep breeds were represented by two ancestral populations in which GKR and AKR were distinct, while KRK and MKR were mixed populations. Genetic distance-based tree, discriminant analysis of principal components (DAPC), and ADMIXTURE analyses confirmed that GKR, which is believed to be derived from the AKR breed, has become genetically different from other sheep populations. Moreover, no migration from and into the GKR breed was detected, while a migration edge was drawn from the KRK and MKR clade into the AKR breed by the TreeMix algorithm. This study confirmed that high-density SNP data obtained from advanced next-generation sequencing (NGS) platforms are efficient in genetically distinguishing animal breeds, even varieties. It is recommended that further NGS-based studies could be carried out to better characterize different indigenous livestock populations raised in Türkiye.

Insights into the abundance, expression and diversity of key denitrification genes in an ecologically managed greenhouse agricultural soil

Understanding the bacteria associated with nitrification and denitrification is crucial for comprehending the processes that lead to nitrous oxide emissions in agricultural greenhouse soils. Therefore, it is important to determine their abundance and expression to gain insight into these processes.The aim of this study was to explore the bacterial communities associated with denitrification in a greenhouse agricultural soil amended with crop residues and manure for six years. For this purpose, we proceeded to detect and quantify the genes nirK and nirS and the gene nosZ through clone library construction, sequencing, phylogenetic analysis, and quantitative polymerase chain reaction (qPCR). Sequence analysis based on the clone library revealed that many of the nirS or nirK genes detected were not closely related to known denitrifier bacteria, but some of the nosZ sequences were related to the genera such as Pseudomonas, Halomonas, and Marinobacter. Furthermore, the qPCR revealed a high abundance of DNA copies in nirK, 6.08 × 109 ± 1.16 × 109, while nirS and nosZ showed lower values, 9.05 × 106 ± 1.65 × 106 and 8.71 × 106 ± 1.44 × 106, respectively. However, the highest expression rate was observed for nirS (mRNA/DNA ratio = 3.10 × 10− 3), while nirK and nosZ showed 10-fold lower expression rates (4.4 × 10− 4 and 3.5 × 10− 4, respectively). The results of this work provide a preliminary overview of the diversity, abundance and expression of key genes associated with the denitrification process in this type of soil and are a starting point for further studies to understand how this type of soil management can influence the denitrification process.

Abstract PO1-15-12: Circulating tumor DNA (ctDNA) for prediction of pathologic complete response (pCR) after 12 weeks of pembrolizumab + trastuzumab + pertuzumab in HER2-enriched early breast cancer: WSG-Keyriched-1 trial

Abstract Background ctDNA testing is emerging as an important biomarker in early breast cancer (eBC). However, its value in prediction of tumor response to de-escalated, chemotherapy-free neoadjuvant therapy (NAT) remains underexplored. In WSG-Keyriched-1 (NCT03988036), a single-arm phase 2 trial, we investigated for the first time a chemotherapy-free NAT with dual HER2 blockade and pembrolizumab in HER2- enriched HER2+ eBC. In this pre-specified translational analysis, we evaluated whether ctDNA measurement could predict pCR. Methods 48 patients (pts) with HER2 2+ (ISH+) or 3+ eBC (stage I-III) and HER2-enriched subtype by PAM50 were included. Pts received pembrolizumab (200 mg), trastuzumab biosimilar (loading dose (LD) 8 mg/kg bodyweight (BW), maintenance dose (MD) 6 mg/kg BW), and pertuzumab (LD 840 mg/kg BW, MD 420 mg/kg BW) q3w for 12 weeks. Primary objective was pCR (ypT0/is ypN0). ctDNA was analyzed in 92 plasma samples collected from 31 pts at baseline (BL) and week 3 of NAT and 30 pts at end of treatment (EOT). Sequencing libraries with unique molecular identifiers were constructed from cell-free DNA and hybridization panels with ≤50 patient-specific somatic mutations from tumor sequencing were used for enrichment. Libraries were sequenced to an ultra-high depth of 100,000×. Sequencing data was analyzed using a combination of public pipelines (megSAP and umiVar2 for mapping and deduplication) and custom in-house scripts (to extract the allele counts for patient-specific variants). To reduce the error rate, only corrected reads with ≥4 duplicates were used. p-value for ctDNA detection was calculated using the total variant count, depth and sample-specific error. Association between ctDNA with pCR and other clinical parameters were assessed with Chi-square or Fisher’s exact test and univariable logistic regression. Additionally, bivariable logistic regressions for pCR were performed with ctDNA and either cT, cN, or grade. Results ctDNA was detected (ctDNA+) in 58.0% (n=18/31) of pts at BL, 9.7% (n=3/31) at week 3, and 10% (n=3/30) at EOT. ctDNA was cleared by week 3 in 83.3% of pts (n=15/18). Compared with ctDNA-negative cases (ctDNA-) at BL, those ctDNA+ more often had cT2-3 disease (94.4%, n=17/18, vs 38.5%, n=5/13; p=.001) and lymph node involvement (61.1%, n=11/18, vs 0%, n=0/13; p< .001). 72.2% (n=13/18) of ctDNA+ and 61.5% (n=8/13) of ctDNA- cases at BL were grade 3 (p=.53). Each of the 3 pts who remained ctDNA+ at week 3 was node-positive; all pts remaining ctDNA- at week 3 were node-negative. pCR rate was 38.9% (n=7/18) in ctDNA+ vs 76.9% (n=10/13) in ctDNA- pts at BL (p=.067), 0% (n=0/3) in ctDNA+ vs 60.7% (n=17/28) in ctDNA- at week 3 (p=.081), and 33.3% (n=1/3) in ctDNA+ vs 59.3% (n=16/27) in ctDNA- at EOT (p=.565). pCR rate was highest in pts who remained ctDNA- throughout week 3 (76.9%, n=10/13), compared to pts with ctDNA cleared by week 3 (46.7%, n=7/15), and pts remaining ctDNA+ (0%, n=0/3, p=.024). ctDNA at BL was predictive for pCR in univariable analysis (odds ratio, OR 0.22, 95%CI 0.04- 0.93, area under curve, AUC=0.69). Bivariable models including ctDNA and grade yielded the highest accuracy for analyses with ctDNA at BL (AUC=0.77), week 3 (AUC=0.79), and with ctDNA change from BL to week 3 (AUC=0.87). Conclusions Absence of ctDNA prior to and during NAT, as well as early clearance of ctDNA associates with a higher pCR rate in eBC pts with HER2-enriched intrinsic subtype treated with chemotherapy-free pembrolizumab plus dual HER2 blockade. These results are in line with prior data with more intensive therapy from I-SPY 2 and NeoALTTO trials in HER2+ eBC and indicate that ctDNA analysis during NAT appears feasible for real- time monitoring of response to treatment and could be used to guide escalation/de-escalation strategies. Further trials with a larger number of pts and pre-designed follow-up are needed to confirm the role of ctDNA for prediction of tumor response and relapse. Citation Format: Monika Graeser, Sherko Kuemmel, Oleg Gluz, Christopher Schroeder, Leon Schuetz, Olga Kelemen, Stephan Ossowski, Katarzyna Jozwiak, Mattea Reinisch, Athina Kostara, Iris Scheffen, Kerstin Lüdtke-Heckenkamp, Felix Hilpert, Angela Kentsch, Carsten Ziske, Reinhard Depenbusch, Michael Braun, Jens-Uwe Blohmer, Matthias Christgen, Stephan Bartels, Hans-Heinrich Kreipe, Enrico Pelz, Ulrike Nitz, Peter Schmid, Nadia Harbeck, Andreas Hartkopf. Circulating tumor DNA (ctDNA) for prediction of pathologic complete response (pCR) after 12 weeks of pembrolizumab + trastuzumab + pertuzumab in HER2-enriched early breast cancer: WSG-Keyriched-1 trial [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-15-12.

Abstract PO3-24-04: Altered epigenetic modifications and genome architecture reshape lipid metabolism during secondary trastuzumab resistance formation of HER2-positive breast cancer

Abstract Title Altered epigenetic modifications and genome architecture reshape lipid metabolism during secondary trastuzumab resistance formation of HER2-positive breast cancer Background Secondary trastuzumab resistance seriously affects the treatment of HER2-positive breast cancer. Although studies have demonstrated several key cellular properties that are closely accompanied with trastuzumab resistance formation, we still have poor knowledge on the genetic and epigenetic variations that promote such transformation. Here we suggested that the epigenetic modification changes on histone H3 together with altered chromatin architecture promote lipid metabolism reprogramming during secondary trastuzumab formation. Materials and methods Induced secondary trastuzumab-resistant SKBR3_HR cell line together with the original trastuzumab-sensitive SKBR3 cell line were applied in this study. Total RNA was collected for transcriptome analysis. Anti-H3K4me3 and K27me3 antibodies were chosen for CUT&Tag sequencing library preparation. Total genome DNA was prepared for Micro-C sequencing library preparation. Activity score of metabolism pathway was calculated as relative gene expression value averaged over all genes in this pathway in certain cell types. Results Upregulation of arachidonic acid metabolism and downregulation of unsaturated fatty acid synthesis, which are mainly characterized by the activation of PTGS1 and PTGES genes and the repression of FASN and SCD genes, respectively, were observed during the formation of secondary trastuzumab resistance from SKBR3 cell to SKBR3_HR cells. Variations of H3k4me3 instead of H3k27me3 regulate the expression of these 4 genes. The accumulation of H3k4me3 was detected at the promoters of PTGS1 and PTGES genes while they were removed from the promoters of FASN and SCD genes in SKBR3_HR cells. More intra-chromosomal interactions were constituted during resistance formation. In detail, 4626 and 4394 topological associated domains, 3125 and 5824 DNA loops were founded in SKBR3 and SKBR3_HR cells, respectively. Furthermore, the lost and gained DNA loops between SCD and PTGS1 genes and distant genome regions may indicate the weaken and strengthen interactions with transcriptional regulators located there. Conclusions During trastuzumab resistance formation, altered histone modifications as well as higher genome structure could regulate the expression of key genes in lipid metabolism pathways, which may further affect cell properties and interactions with cells in tumor microenvironment. Citation Format: Ningjun Duan, Yijia Hua, Yongmei Yin. Altered epigenetic modifications and genome architecture reshape lipid metabolism during secondary trastuzumab resistance formation of HER2-positive breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-24-04.

RNA sequencing methods enable immune cell gene expression profiling and clonality determination

Abstract The adaptive immune system defends the human body from pathogens through B/T cell functions in various organs. Abnormality of these cells may reflect immunological disorder, presence of neoplastic tissue, or response to infectious agents. High-throughput sequencing technology has facilitated understanding gene expression and related cell functions. Still, an in-depth understanding of B and T cell phenotype and clonotype profiling is an emerging application that has yet to take full advantage of sequencing technology for disease diagnosis and prognosis. Here, we share results of RNA sequencing of immune system tissues, including healthy donor peripheral blood mononuclear cells (PBMC), lymph nodes, bone marrow, spleen, thyroid, thymus, and colon, as well as diseased PBMC and colon samples. Sequencing libraries were constructed and sequenced for B and T cell-specific gene expression and full-length immune repertoire profiling. We have identified tissue-specific B/T cell phenotype and clonality patterns related to tissue functions and disease progression. RNA-seq enables highly multiplex immunophenotyping and clonality determination for high-abundant clones that correlate with Immune-seq, with the latter targeted approach providing more in-depth clonality signatures. Integrating both RNA sequencing and immune repertoire sequencing approaches allows accurate phenotype and clonotype determination for the immune landscape in various tissues.

DengueSeq: a pan-serotype whole genome amplicon sequencing protocol for dengue virus

BackgroundThe increasing burden of dengue virus on public health due to more explosive and frequent outbreaks highlights the need for improved surveillance and control. Genomic surveillance of dengue virus not only provides important insights into the emergence and spread of genetically diverse serotypes and genotypes, but it is also critical to monitor the effectiveness of newly implemented control strategies. Here, we present DengueSeq, an amplicon sequencing protocol, which enables whole-genome sequencing of all four dengue virus serotypes.ResultsWe developed primer schemes for the four dengue virus serotypes, which can be combined into a pan-serotype approach. We validated both approaches using genetically diverse virus stocks and clinical specimens that contained a range of virus copies. High genome coverage (>95%) was achieved for all genotypes, except DENV2 (genotype VI) and DENV 4 (genotype IV) sylvatics, with similar performance of the serotype-specific and pan-serotype approaches. The limit of detection to reach 70% coverage was 10-100 RNA copies/μL for all four serotypes, which is similar to other commonly used primer schemes. DengueSeq facilitates the sequencing of samples without known serotypes, allows the detection of multiple serotypes in the same sample, and can be used with a variety of library prep kits and sequencing instruments.ConclusionsDengueSeq was systematically evaluated with virus stocks and clinical specimens spanning the genetic diversity within each of the four dengue virus serotypes. The primer schemes can be plugged into existing amplicon sequencing workflows to facilitate the global need for expanded dengue virus genomic surveillance.

MORPHOMETRIC CHARACTERIZATION AND THE FIRST TAXONOMIC SYNTHESIS OF TERRESTRIAL CYANOBACTERIA FROM THE ATLANTIC FOREST IN NORTHEASTERN BRAZIL

Cyanobacteria are very morphological diverse and the taxonomic inventories are the basis of the knowledge about their biodiversity. However, these studies have decreased in the last years, mainly due to advances in molecular techniques, evenness these tools have their limitations, like sequence libraries. In order to reveal the importance of taxonomic inventories to little known organisms and unexplored areas, the present study was carried out with terrestrial cyanobacteria from the Atlantic Forest of northeastern Brazil, compared to results with the state of the art of this group in the country. A total of 60 infrageneric taxa were found and 62.8% of identified species were new records to northeastern Brazil. Most of the previous studies were performed in the state of São Paulo (>70%) and half of the country has no records of terrestrial cyanobacteria due to the lack of studies. Thus, taxonomic surveys are still important to understand the cyanobacterial distribution and to characterize rare taxa and the biodiversity from unstudied areas and habitats.

NKX2-2 based nuclei sorting on frozen human archival pancreas enables the enrichment of islet endocrine populations for single-nucleus RNA sequencing

BackgroundCurrent approaches to profile the single-cell transcriptomics of human pancreatic endocrine cells almost exclusively rely on freshly isolated islets. However, human islets are limited in availability. Furthermore, the extensive processing steps during islet isolation and subsequent single cell dissolution might alter gene expressions. In this work, we report the development of a single-nucleus RNA sequencing (snRNA-seq) approach with targeted islet cell enrichment for endocrine-population focused transcriptomic profiling using frozen archival pancreatic tissues without islet isolation.ResultsWe cross-compared five nuclei isolation protocols and selected the citric acid method as the best strategy to isolate nuclei with high RNA integrity and low cytoplasmic contamination from frozen archival human pancreata. We innovated fluorescence-activated nuclei sorting based on the positive signal of NKX2-2 antibody to enrich nuclei of the endocrine population from the entire nuclei pool of the pancreas. Our sample preparation procedure generated high-quality single-nucleus gene-expression libraries while preserving the endocrine population diversity. In comparison with single-cell RNA sequencing (scRNA-seq) library generated with live cells from freshly isolated human islets, the snRNA-seq library displayed comparable endocrine cellular composition and cell type signature gene expression. However, between these two types of libraries, differential enrichments of transcripts belonging to different functional classes could be observed.ConclusionsOur work fills a technological gap and helps to unleash frozen archival pancreatic tissues for molecular profiling targeting the endocrine population. This study opens doors to retrospective mappings of endocrine cell dynamics in pancreatic tissues of complex histopathology. We expect that our protocol is applicable to enrich nuclei for transcriptomics studies from various populations in different types of frozen archival tissues.

Analysis of the characteristics of intestinal microbiota after oral tolerance in infants with food protein-induced proctocolitis.

To understand the characteristics of the intestinal microbiota after oral tolerance in infants with food protein-induced proctocolitis (FPIAP) treated with amino acid formula and their differences from healthy children, aiming to provide a scientific basis for guiding the application of probiotics during treatment. FPIAP infants were prospectively enrolled, fecal specimens were obtained, and DNA was extracted for PCR amplification of the bacterial 16S rRNA gene V4 region. Library construction and sequencing were performed, and bioinformatic analysis was performed after obtaining valid data. There were 36 patients in the FPIAP group: 20 males and 16 females, age 21.944 ± 13.277 months. Diarrhea with blood in the stool were the main symptom, with an average course of 14.83 ± 9.33 days. Thirty infants (83.33%) had mucus stool, 11.11% (4/36) of them experiencing vomiting, and 55.56% (20/36) of the infants displaying poor intake and weight gain, 28 (77.78%) patients with moderate eczema, 2 (5.6%) patients with chronic respiratory symptoms. The treatment time with amino acid formula was 5.51 ± 2.88 months. A control group comprising of 25 healthy infants who were full-term, natural delivery, bottle fed, and matched in terms of age (24.840 ± 12.680 months) and gender (15 males and 10 females) was selected. Anaerobic bacteria were less abundant in FPIAP infants than healthy infants (P = 4.811 × 10-5), but potentially pathogenic bacteria were more abundant (P = 0.000). The abundance of Actinobacteria was low in FPIAP infants, the abundance of Proteobacteria was high, and the abundance of Firmicutes was reduced. Bifidobacterium could be used as a bacterial genus to differentiate healthy and FPIAP infants. Both α-and β-diversity indicators of intestinal microbiota were lower in FPIAP infants. In FPIAP infants, glucose and energy metabolism and amino acid anabolism were decreased, and inflammation-related lipopolysaccharide synthesis pathways were increased. Compared with healthy infants, FPIAP infants with oral tolerance after amino acid formula treatment had differences in the structure and diversity of intestinal microbiota, among which Bifidobacterium was significantly reduced. This trial was registered on https://register.clinicaltrials.gov/.

A PCR-independent approach for mtDNA enrichment and next-generation sequencing: comprehensive evaluation and clinical application

BackgroundSequencing the mitochondrial genome has been increasingly important for the investigation of primary mitochondrial diseases (PMD) and mitochondrial genetics. To overcome the limitations originating from PCR-based mtDNA enrichment, we set out to develop and evaluate a PCR-independent approach in this study, named Pime-Seq (PCR-independent mtDNA enrichment and next generation Sequencing).ResultsBy using the optimized mtDNA enrichment procedure, the mtDNA reads ratio reached 88.0 ± 7.9% in the sequencing library when applied on human PBMC samples. We found the variants called by Pime-Seq were highly consistent among technical repeats. To evaluate the accuracy and reliability of this method, we compared Pime-Seq with lrPCR based NGS by performing both methods simultaneously on 45 samples, yielding 1677 concordant variants, as well as 146 discordant variants with low-level heteroplasmic fraction, in which Pime-Seq showed higher reliability. Furthermore, we applied Pime-Seq on 4 samples of PMD patients retrospectively, and successfully detected all the pathogenic mtDNA variants. In addition, we performed a prospective study on 192 apparently healthy pregnant women during prenatal screening, in which Pime-Seq identified pathogenic mtDNA variants in 4 samples, providing extra information for better health monitoring in these cases.ConclusionsPime-Seq can obtain highly enriched mtDNA in a PCR-independent manner for high quality and reliable mtDNA deep-sequencing, which provides us an effective and promising tool for detecting mtDNA variants for both clinical and research purposes.

MiRNome Profiling Analysis Reveals Novel Hepatocellular Carcinoma Diagnostic, Prognostic and Treatment-Related Candidate Biomarkers: Post hoc Analysis of SORAMIC Trial

Introduction: Early diagnosis of hepatocellular carcinoma (HCC) as well as evaluation of prognosis and prediction of treatment efficacy remains challenging due to the missing specific non-invasive biomarkers. The aim of this study was to identify disease-specific microRNA (miRNA) patterns for diagnosis, prediction of prognosis, and treatment response in patients with HCC. Methods: The study population included 42 HCC patients from SORAMIC clinical trial: 22 patients received sorafenib monotherapy, 20 patients underwent 90Y radioembolization in combination with sorafenib. 20 individuals were included in the control group. HCC patients underwent collection of plasma samples before and 7–9 weeks after the beginning of the treatment. Isolation of circulating miRNAs, preparation of small RNA sequencing libraries and next-generation sequencing were performed. Association analysis for novel diagnostic, prognostic, and treatment-related candidate biomarkers was performed. Results: A total of 42 differentially expressed (16 up-regulated and 26 down-regulated) miRNAs were identified comparing baseline and control group plasma samples. hsa-miR-215-5p and hsa-miR-192-5p were down-regulated, while hsa-miR-483-5p and hsa-miR-23b-3p were up-regulated comparing baseline and 7–9 weeks post-sorafenib monotherapy samples. hsa-miR-215-5p was the sole down-regulated miRNA in the same combination therapy comparison. hsa-miR-183-5p, hsa-miR-28-3p, and hsa-miR-1246 were found to be significantly up-regulated comparing non-responders versus responders to sorafenib. High hsa-miR-215-5p expression was significantly associated with worse HCC patients’ prognosis. Conclusions: Systematic miRNA profiling of highly characterized samples from SORAMIC study revealed a subset of potential miRNA biomarkers for HCC diagnosis and prognosis of sorafenib-treated patients’ survival.