Massively Parallel Sequencing Research Articles

Optical genome mapping unveils hidden structural variants in neurodevelopmental disorders.

While short-read sequencing currently dominates genetic research and diagnostics, it frequently falls short of capturing certain structural variants (SVs), which are often implicated in the etiology of neurodevelopmental disorders (NDDs). Optical genome mapping (OGM) is an innovative technique capable of capturing SVs that are undetectable or challenging-to-detect via short-read methods. This study aimed to investigate NDDs using OGM, specifically focusing on cases that remained unsolved after standard exome sequencing. OGM was performed in 47 families using ultra-high molecular weight DNA. Single-molecule maps were assembled de novo, followed by SV and copy number variant calling. We identified 7 variants of interest, of which 5 (10.6%) were classified as likely pathogenic or pathogenic, located in BCL11A, OPHN1, PHF8, SON, and NFIA. We also identified an inversion disrupting NAALADL2, a gene which previously was found to harbor complex rearrangements in two NDD cases. Variants in known NDD genes or candidate variants of interest missed by exome sequencing mainly consisted of larger insertions (> 1kbp), inversions, and deletions/duplications of a low number of exons (1-4 exons). In conclusion, in addition to improving molecular diagnosis in NDDs, this technique may also reveal novel NDD genes which may harbor complex SVs often missed by standard sequencing techniques.

Long-read powered viral metagenomics in the oligotrophic Sargasso Sea

Dominant microorganisms of the Sargasso Sea are key drivers of the global carbon cycle. However, associated viruses that shape microbial community structure and function are not well characterised. Here, we combined short and long read sequencing to survey Sargasso Sea phage communities in virus- and cellular fractions at viral maximum (80 m) and mesopelagic (200 m) depths. We identified 2,301 Sargasso Sea phage populations from 186 genera. Over half of the phage populations identified here lacked representation in global ocean viral metagenomes, whilst 177 of the 186 identified genera lacked representation in genomic databases of phage isolates. Viral fraction and cell-associated viral communities were decoupled, indicating viral turnover occurred across periods longer than the sampling period of three days. Inclusion of long-read data was critical for capturing the breadth of viral diversity. Phage isolates that infect the dominant bacterial taxa Prochlorococcus and Pelagibacter, usually regarded as cosmopolitan and abundant, were poorly represented.

Disentangling oncogenic amplicons in esophageal adenocarcinoma

Esophageal adenocarcinoma is a prominent example of cancer characterized by frequent amplifications in oncogenes. However, the mechanisms leading to amplicons that involve breakage-fusion-bridge cycles and extrachromosomal DNA are poorly understood. Here, we use 710 esophageal adenocarcinoma cases with matched samples and patient-derived organoids to disentangle complex amplicons and their associated mechanisms. Short-read sequencing identifies ERBB2, MYC, MDM2, and HMGA2 as the most frequent oncogenes amplified in extrachromosomal DNAs. We resolve complex extrachromosomal DNA and breakage-fusion-bridge cycles amplicons by integrating of de-novo assemblies and DNA methylation in nine long-read sequenced cases. Complex amplicons shared between precancerous biopsy and late-stage tumor, an enrichment of putative enhancer elements and mobile element insertions are potential drivers of complex amplicons’ origin. We find that patient-derived organoids recapitulate extrachromosomal DNA observed in the primary tumors and single-cell DNA sequencing capture extrachromosomal DNA-driven clonal dynamics across passages. Prospectively, long-read and single-cell DNA sequencing technologies can lead to better prediction of clonal evolution in esophageal adenocarcinoma.

Результаты анализа состава микрофлоры рубца коров под влиянием пробиотика методом NGS-секвенирования

The purpose of research is to study the effect of the probiotic Profort-T on the composition of the rumen microbiome of dairy cows using NGS sequencing, productivity and hematological parameters of the animals' blood serum. The experiment was carried out on black-and-white cows for 60 days. Analogue groups of 20 animals each were formed: control group I (received the basic diet (BD)) and experimental group II (received the BD diet and the Profort-T probiotic). The BD consisted of: silage – 42.26 kg; hay – 1.97; mixed feed – 14.67 kg; salt, chalk, soda. The cows of experimental group II received the probiotic Profort-T at the rate of 0.03 kg per 1 head from the day of admission to the new calving group. The feed additive was distributed manually to each head of experimental group II during the new calving period and partially during milking. The animals were kept under the same conditions. Animals are kept in tethered conditions. The rumen bacterial community was assessed by NGS sequencing using a MiSeq sequencer (Illumina, Inc., USA). The use of a probiotic (experimental group II) made it possible to increase the average daily milk yield from 14.9 to 22.0 % compared to the control (P ≤ 0.05) and reduce the content of somatic cells in cows’ milk from 21.6 to 33.5 % (P ≤ 0.05). NGS sequencing demonstrated that 21 phyla of microorga¬nisms were found in the rumen microflora of the studied cows. The most represented (P ≤ 0.05) bacteria in the rumen were bacteria of the phylum Bacteroidetes – from 46.6 ± 4.48 to 58.1 ± 5.52 %. The proportion of cellulolytic bacteria Candidatus Saccharibacteria increased in experimental group II at the end of the experiment compared to the control by 1.9 times (P ≤ 0.05). Microorganisms Proteobacteria and Fusobacteria decreased in experimental group II compared to the control by 5.7 and 1.8 times, respectively (P ≤ 0.05), i.e. the use of the Profort-T probiotic led to an improvement in the structure of the microbial community of the cow rumen.

RNA-Seq transcriptome profiling of immature grain wheat is a technique for understanding comparative modeling of baking quality

Improving the baking quality is a primary challenge in the wheat flour production value chain, as baking quality represents a crucial factor in determining its overall value. In the present study, we conducted a comparative RNA-Seq analysis on the high baking quality mutant “O-64.1.10” genotype and its low baking quality wild type "Omid" cultivar to recognize potential genes associated with bread quality. The cDNA libraries were constructed from immature grains that were 15 days post-anthesis, with an average of 16.24 and 18.97 million paired-end short-read sequences in the mutant and wild-type, respectively. A total number of 733 transcripts with differential expression were identified, 585 genes up-regulated and 188 genes down-regulated in the “O-64.1.10” genotype compared to the “Omid”. In addition, the families of HSF, bZIP, C2C2-Dof, B3-ARF, BES1, C3H, GRF, HB-HD-ZIP, PLATZ, MADS-MIKC, GARP-G2-like, NAC, OFP and TUB were appeared as the key transcription factors with specific expression in the “O-64.1.10” genotype. At the same time, pathways related to baking quality were identified through Kyoto Encyclopedia of Genes and Genomes. Collectively, we found that the endoplasmic network, metabolic pathways, secondary metabolite biosynthesis, hormone signaling pathway, B group vitamins, protein pathways, pathways associated with carbohydrate and fat metabolism, as well as the biosynthesis and metabolism of various amino acids, have a great deal of potential to play a significant role in the baking quality. Ultimately, the RNA-seq results were confirmed using quantitative Reverse Transcription PCR for some hub genes such as alpha-gliadin, low molecular weight glutenin subunit and terpene synthase (gibberellin) and as a resource for future study, 127 EST-SSR primers were generated using RNA-seq data.

A set of nematode rRNA cistron databases and a primer assessment tool to enable more flexible and comprehensive metabarcoding.

The ITS-2-rRNA has been particularly useful for nematode metabarcoding but does not resolve all phylogenetic relationships, and reference sequences are not available for many nematode species. This is a particular issue when metabarcoding complex communities such as wildlife parasites or terrestrial and aquatic free-living nematode communities. We have used markerDB to produce four databases of distinct regions of the rRNA cistron: the 18S rRNA gene, the 28S rRNA gene, the ITS-1 intergenic spacer and the region spanning ITS-1_5.8S_ITS-2. These databases comprise 2645, 254, 13,461 and 10,107 unique full-length sequences representing 1391, 204, 1837 and 1322 nematode species, respectively. The comparative analysis illustrates the complementary value but also reveals a better representation of Clade III, IV and V than Clade I and Clade II nematodes in each case. Although the ITS-1 database includes the largest number of unique full-length sequences, the 18S rRNA database provides the widest taxonomic coverage. We also developed PrimerTC, a tool to assess primer sequence conservation across any reference sequence database, and have applied it to evaluate a large number of previously published rRNA cistron primers. We identified sets of primers that currently provide the broadest taxonomic coverage for each rRNA marker across the nematode phylum. These new resources will facilitate more comprehensive metabarcoding of nematode communities using either short-read or long-read sequencing platforms. Further, PrimerTC is available as a simple WebApp to guide or assess PCR primer design for any genetic marker and/or taxonomic group beyond the nematode phylum.

Machine learning and metagenomics identifies uncharacterized taxa inferred to drive biogeochemical cycles in a subtropical hypereutrophic estuary

Abstract Anthropogenic influences have drastically increased nutrient concentrations in many estuaries globally, and microbial communities have adapted to the resulting hypereutrophic ecosystems. However, our knowledge of the dominant microbial taxa and their potential functions in these ecosystems has remained sparse. Here, we study prokaryotic community dynamics in a temporal–spatial dataset, from a subtropical hypereutrophic estuary. Screening 54 water samples across brackish to marine sites revealed that nutrient concentrations and salinity best explained spatial community variations, whereas temperature and dissolved oxygen likely drive seasonal shifts. By combining short and long read sequencing data, we recovered 2459 metagenome-assembled genomes (MAGs), proposed new taxon names for previously uncharacterised lineages, and created an extensive, habitat specific genome reference database. Community profiling based on this genome reference database revealed a diverse prokaryotic community comprising 61 bacterial and 18 archaeal phyla, and resulted in an improved taxonomic resolution at lower ranks down to genus level. We found that the vast majority (61 out of 73) of abundant genus level taxa (>1% average) represented unnamed and novel lineages, and that all genera could be clearly separated into brackish and marine ecotypes with inferred habitat specific functions. Applying supervised machine learning and metabolic reconstruction, we identified several microbial indicator taxa responding directly or indirectly to elevated nitrate and total phosphorus concentrations. In conclusion, our analysis highlights the importance of improved taxonomic resolution, sheds light on the role of previously uncharacterised lineages in estuarine nutrient cycling, and identifies microbial indicators for nutrient levels crucial in estuary health assessments.

Co-occurrence of triple carbapenemase genes, bla VIM-2, bla NDM-1, and bla OXA-48 in Enterobacter hormaechei clinical isolates –first report from Croatia

Two Enterobacter hormaechei isolates harbouring three carbapenemase genes each, were isolated from two patients from different ICUs at University Hospital Centre Zagreb, Croatia, which is to our knowledge, the first report of triple carbapenemase (bla VIM-2, bla NDM-1, and bla OXA-48 ) co-existence in E. hormachei strains and also among Enterobacterales members in Croatia. Antimicrobial susceptibility testing showed susceptibility only to colistin and amikacin. The production of carbapenemases was phenotypically tested by immunochromatographic assay and confirmed by PCR. Detailed analysis by Whole Genome Sequencing (WGS) of short reads by Illumina and long reads by Oxford Nanopore Technologies (ONT) was additionally performed and showed that both isolates belonged to ST200. They were separated by 98 Single Nucleotide Polymorphisms (SNPs) having variations in the number of bla VIM-2 genes on the chromosome, the number of bla NDM-1 genes on the plasmid, non-identical bla NDM-1 plasmids, different plasmid content in general, and only one isolate carried a 94 kb prophage.

A comparison between full-length 16S rRNA Oxford nanopore sequencing and Illumina V3-V4 16S rRNA sequencing in head and neck cancer tissues.

Describing the microbial community within the tumour has been a key aspect in understanding the pathophysiology of the tumour microenvironment. In head and neck cancer (HNC), most studies on tissue samples have only performed 16S rRNA short-read sequencing (SRS) on V3-V5 region. SRS is mostly limited to genus level identification. In this study, we compared full-length 16S rRNA long-read sequencing (FL-ONT) from Oxford Nanopore Technology (ONT) to V3-V4 Illumina SRS (V3V4-Illumina) in 26 HNC tumour tissues. Further validation was also performed using culture-based methods in 16 bacterial isolates obtained from 4 patients using MALDI-TOF MS. We observed similar alpha diversity indexes between FL-ONT and V3V4-Illumina. However, beta-diversity was significantly different between techniques (PERMANOVA - R2 = 0.131, p < 0.0001). At higher taxonomic levels (Phylum to Family), all metrics were more similar among sequencing techniques, while lower taxonomy displayed more discrepancies. At higher taxonomic levels, correlation in relative abundance from FL-ONT and V3V4-Illumina were higher, while this correlation decreased at lower levels. Finally, FL-ONT was able to identify more isolates at the species level that were identified using MALDI-TOF MS (75% vs. 18.8%). FL-ONT was able to identify lower taxonomic levels at a better resolution as compared to V3V4-Illumina 16S rRNA sequencing.

Evaluation of the accuracy of bacterial genome reconstruction with Oxford Nanopore R10.4.1 long-read-only sequencing.

Whole-genome reconstruction of bacterial pathogens has become an important tool for tracking transmission and antimicrobial resistance gene spread, but highly accurate and complete assemblies have largely only historically been achievable using hybrid long- and short-read sequencing. We previously found the Oxford Nanopore Technologies (ONT) R10.4/kit12 flowcell/chemistry produced improved assemblies over the R9.4.1/kit10 combination, however long-read only assemblies contained more errors compared to Illumina-ONT hybrid assemblies. ONT have since released an R10.4.1/kit14 flowcell/chemistry upgrade and recommended the use of Bovine Serum Albumin (BSA) during library preparation, both of which reportedly increase accuracy and yield. They have also released updated basecallers trained using native bacterial DNA containing methylation sites intended to fix systematic basecalling errors, including common adenosine (A) to guanine (G) and cytosine (C) to thymine (T) substitutions. To evaluate these improvements, we successfully sequenced four bacterial reference strains, namely Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus, and nine genetically diverse E. coli bloodstream infection-associated isolates from different phylogroups and sequence types, both with and without BSA. These sequences were de novo assembled and compared against Illumina-corrected reference genomes. In this small evaluation of 13 isolates we found that nanopore long-read-only R10.4.1/kit 14 assemblies with updated basecallers trained using bacterial methylated DNA produce accurate assemblies with ≥40×depth, sufficient to be cost-effective compared with hybrid ONT/Illumina sequencing in our setting.

Demographic fluctuations in bloodstream Staphylococcus aureus lineages configure the mobile gene pool and antimicrobial resistance

Staphylococcus aureus in the bloodstream causes high morbidity and mortality, exacerbated by the spread of multidrug-resistant and methicillin-resistant S. aureus (MRSA). We aimed to characterize the circulating lineages of S. aureus from bloodstream infections and the contribution of individual lineages to resistance over time. Here, we generated 852 high-quality short-read draft genome sequences of S. aureus isolates from patient blood cultures in a single hospital from 2010 to 2022. A total of 80 previously recognized sequence types (ST) and five major clonal complexes are present in the population. Two frequently detected lineages, ST5 and ST8 exhibited fluctuating demographic structures throughout their histories. The rise and fall in their population growth coincided with the acquisition of antimicrobial resistance, mobile genetic elements, and superantigen genes, thus shaping the accessory genome structure across the entire population. These results reflect undetected selective events and changing ecology of multidrug-resistant S. aureus in the bloodstream.

Emergence of an IncX3 plasmid co-harbouring the carbapenemase genes blaNDM-5 and blaOXA-181.

The spread of transmissible plasmids with carbapenemase genes has contributed to a global increase in carbapenemase-producing Enterobacterales over the past two decades, with blaNDM and blaOXA among the most prevalent carbapenemase genes. To characterize an Escherichia coli isolate co-carrying blaNDM-5 and blaOXA-181 (JBEHAAB-19-0176) that was isolated in the Japan Antimicrobial Resistant Bacterial Surveillance in 2019-20, and to evaluate the functional advantage of carrying both genes as opposed to only one. The whole-genome sequence of the isolate was determined using long- and short-read sequencing. Growth assay and co-culture experiments were performed for phenotypic characterization in the presence of different β-lactam antibiotics. WGS analysis showed that blaNDM-5 and blaOXA-181 were carried by the same IncX3 plasmid, pJBEHAAB-19-0176_NDM-OXA. Genetic characterization of the plasmid suggested that the plasmid emerged through the formation of a co-integrate and resolution of two typical IncX3 plasmids harbouring blaNDM-5 and blaOXA-181, which involved two recombination events at the IS3000 and IS26 sequences. When cultured in the presence of piperacillin or cefpodoxime, the growth rate of the transformant co-harbouring blaNDM-5 and blaOXA-181 was significantly higher than the transformant with only blaNDM-5. Furthermore, in co-culture where the two blaNDM-5-harbouring transformants were allowed to compete directly, the strain additionally harbouring blaOXA-181 showed a marked growth advantage. The additional carriage of blaOXA-181 confers a selective advantage to bacteria in the presence of piperacillin and cefpodoxime. These findings may explain the current epidemiology of carbapenemase-producing Enterobacterales, in which bacteria carrying both blaNDM-5 and blaOXA-48-like genes have emerged independently worldwide.

Redefining the bacteriophage mv4 site-specific recombination system and the sequence specificity of its attB and core-attP sites.

Through their involvement in the integration and excision of a large number of mobile genetic elements, such as phages and integrative and conjugative elements (ICEs), site-specific recombination systems based on heterobivalent tyrosine recombinases play a major role in genome dynamics and evolution. However, despite hundreds of these systems having been identified in genome databases, very few have been described in detail, with none from phages that infect Bacillota (formerly Firmicutes). In this study, we reanalyzed the recombination module of Lactobacillus delbrueckii subsp. bulgaricus phage mv4, previously considered atypical compared with classical systems. Our results reveal that mv4 integrase is a 369 aa protein with all the structural hallmarks of recombinases from the Tn916 family and that it cooperatively interacts with its recombination sites. Using randomized DNA libraries, NGS sequencing, and other molecular approaches, we show that the 21-bp core-attP and attB sites have structural similarities to classical systems only if considering the nucleotide degeneracy, with two 7-bp inverted regions corresponding to mv4Int core-binding sites surrounding a 7-bp strand-exchange region. We also examined the different compositional constraints in the core-binding regions, which define the sequence space of permissible recombination sites.

Long-read sequencing improves the genetic diagnosis of retinitis pigmentosa by identifying an Alu retrotransposon insertion in the EYS gene

BackgroundBiallelic variants in EYS are the major cause of autosomal recessive retinitis pigmentosa (arRP) in certain populations, a clinically and genetically heterogeneous disease that may lead to legal blindness. EYS is one of the largest genes (~ 2 Mb) expressed in the retina, in which structural variants (SVs) represent a common cause of disease. However, their identification using short-read sequencing (SRS) is not always feasible. Here, we conducted targeted long-read sequencing (T-LRS) using adaptive sampling of EYS on the MinION sequencing platform (Oxford Nanopore Technologies) to definitively diagnose an arRP family, whose affected individuals (n = 3) carried the heterozygous pathogenic deletion of exons 32–33 in the EYS gene. As this was a recurrent variant identified in three additional families in our cohort, we also aimed to characterize the known deletion at the nucleotide level to assess a possible founder effect.ResultsT-LRS in family A unveiled a heterozygous AluYa5 insertion in the coding exon 43 of EYS (chr6(GRCh37):g.64430524_64430525ins352), which segregated with the disease in compound heterozygosity with the previously identified deletion. Visual inspection of previous SRS alignments using IGV revealed several reads containing soft-clipped bases, accompanied by a slight drop in coverage at the Alu insertion site. This prompted us to develop a simplified program using grep command to investigate the recurrence of this variant in our cohort from SRS data. Moreover, LRS also allowed the characterization of the CNV as a ~ 56.4kb deletion spanning exons 32–33 of EYS (chr6(GRCh37):g.64764235_64820592del). The results of further characterization by Sanger sequencing and linkage analysis in the four families were consistent with a founder variant.ConclusionsTo our knowledge, this is the first report of a mobile element insertion into the coding sequence of EYS, as a likely cause of arRP in a family. Our study highlights the value of LRS technology in characterizing and identifying hidden pathogenic SVs, such as retrotransposon insertions, whose contribution to the etiopathogenesis of rare diseases may be underestimated.

The phased Solanum okadae genome and Petota pangenome analysis of 23 other potato wild relatives and hybrids

Potato is an important crop in the genus Solanum section Petota. Potatoes are susceptible to multiple abiotic and biotic stresses and have undergone constant improvement through breeding programs worldwide. Introgression of wild relatives from section Petota with potato is used as a strategy to enhance the diversity of potato germplasm. The current dataset contributes a phased genome assembly for diploid S. okadae, and short read sequences and de novo assemblies for the genomes of 16 additional wild diploid species in section Petota that were noted for stress resistance and were of interest to potato breeders. Genome sequence data for three additional genomes representing polyploid hybrids with cultivated potato, and an additional genome from non-tuberizing S. etuberosum, which is outside of section Petota, were also included. High quality short reads assemblies were achieved with genome sizes ranging from 575 to 795 Mbp and annotations were performed utilizing transcriptome sequence data. Genomes were compared for presence/absence of genes and phylogenetic analyses were carried out using plastome and nuclear sequences.

An overview of autosomal STRs and identity SNPs in a Norwegian population using massively parallel sequencing

In recent years, probabilistic genotyping software has been adapted for the analysis of massively parallel sequencing (MPS) forensic data. Likelihood ratios (LR) are based on allele frequencies selected from populations of interest. This study provides an outline of sequence-based (SB) allele frequencies for autosomal short tandem repeats (aSTRs) and identity single nucleotide polymorphisms (iSNPs) in 371 individuals from Southern Norway. 27 aSTRs and 94 iSNPs were previously analysed with the ForenSeq™ DNA Signature Prep Kit (Verogen). The number of alleles with frequencies less than 0.05 for sequenced-based alleles was 4.6 times higher than for length-based alleles. Consistent with previous studies, it was observed that sequence-based data (both with and without flanks) exhibited higher allele diversity compared to length-based (LB) data; random match probabilities were lower for SB alleles confirming their advantage to discriminate between individuals. Two alleles in markers D22S1045 and Penta D were observed with SNPs in the 3´ flanking region, which have not been reported before. Also, a novel SNP with a minor allele frequency (MAF) of 0.001, was found in marker TH01. The impact of the sample size on minor allele frequency (MAF) values was studied in 88 iSNPs from Southern Norway (n = 371). The findings were then compared to a larger Norwegian population dataset (n = 15,769). The results showed that the smaller Southern Norway dataset provided similar results, and it was a representative sample. Population structure was analyzed for regions within Southern Norway; FST estimates for aSTR and iSNPs did not indicate any genetic structure. Finally, we investigated the genetic differences between Southern Norway and two other populations: Northern Norway and Denmark. Allele frequencies between these populations were compared, and we found no significant frequency differences (p-values > 0.0001). We also calculated the pairwise FST values per marker and comparisons between Southern and Northern Norway showed small differences. In contrast, the comparisons between Southern Norway and Denmark showed higher FST values for some markers, possibly driven by distinct alleles that were present in only one of the populations. In summary, we propose that allele frequencies from each population considered in this study could be used interchangeably to calculate genotype probabilities.

Abstract PO4-08-08: Is it time for a European BRCA Mutation Registry? An Italian experience

Abstract Background: Knowledge of BRCA 1/2 mutation plays an important role in cancer care. The National Plan to improve BRCA detection in order to increase access to target agents and ameliorate the prevention strategy has several implementations in the Italian regions. Recent guidelines are considering extending genetic testing to a larger population, regardless of family history. We present our experience in Apulia Region on the detection of BRCA 1/2 and its clinical implication at the regional level, to underline the importance of spreading the extension of BRCA detection at the regional level and to propose the creation of a European register for BRCA patients, based on population-based BRCA 1/2 testing. Methods: This is an observational study conducted at a Familiar Cancer Service of the Lecce Hospital. Participants completed a questionnaire (socio-demographic epidemiology of cancer and lifestyle factors), genetic counseling and BRCA testing. Notably, genetic testing for BRCA mutations was carried out based on family cancer history since 2018, according to the Italian cancer guidelines (AIOM) and related updates in 2019 and 2023. A mutational analysis of exons and adjacent intronic regions of BRCA1/BRCA2 genes was performed by Sanger sequencing and MiSeq Illumina NGS platform. Results: Between 2014 and May 2023 a total of 4365 patients were enrolled in the study, of whom 2426 (56%) was evaluable for BRCA detection, 18% men (n=440) and 82% female (n=1986). Median age at diagnosis was 57 yrs. 65% of patients had a cancer diagnosis (n=1575) and 35% were healthy carriers (n=851). In particular, breast cancer (BC) was present in 63% of patients (n=1048), ovarian cancer (OC) in 21% (n=352), pancreatic cancer in 5% (n=85), prostate cancer in 4% (n=65), melanoma in 1% (n=13), and other cancers in 6% (n=92). The table 1 (uploaded) shows the results of patients with BC (n=1048) The presence of BRCA mutation is observed in 20.52% of BC patients (1:4.87). Conclusion These data confirm that BRCA mutation detection is an emerging need to improve prevention and early diagnosis according to the European Beating Cancer Plan. Therefore, it is crucial to improve this topic at regional level until the creation of a shared BRCA registry among European countries. Table. Citation Format: Elisabetta De Matteis, Mariangela Ciccarese, Paolo Tarantino, Tiziana Grassi, Francesco Bagordo, Emanuele Rizzo, Maria Rosaria Tumolo, Graziana Ronzino, Maria Rita De Giorgio, Salvatore Mauro, Nicola Di Renzo. Is it time for a European BRCA Mutation Registry? An Italian experience [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 PO4-08-08.

Abstract PO4-01-04: Clinical significance of Circulating tumor DNA (ctDNA) analysis in predicting pathologic complete remission in HER2-positive Breast Cancer Patients Undergoing Neoadjuvant Chemotherapy

Abstract Background: Neoadjuvant chemotherapy (NACT) combined with dual anti-HER2 agents followed by surgical resection is the standard treatment for stage II-III HER2-positive breast cancer. Despite the availability of effective therapies as an adjuvant treatment such as ado-trastuzumab emtansine (T-DM1) and neratinib, pathologic complete remission (pCR) strongly predicts survival. This study aimed to investigate the role of ctDNA analysis in predicting pCR and clinical outcome using comprehensive ctDNA analysis. Methods: A total of 34 HER2-positive breast cancer patients who underwent NACT combined with dual anti-HER2 agents and surgical resection were enrolled, including 22 clinical stage II and 12 III patients. Plasma samples were collected at four-time points: before neoadjuvant chemotherapy, after 3 cycles, at the completion of neoadjuvant chemotherapy, and after surgery. low-pass whole-genome sequencing (lpWGS) and whole exome sequencing (WES) in evaluating blood copy-number burden (bCNB), genomic alterations, mutational signatures, blood tumor mutational burden (bTMB). Mutations selected from the baseline sample were monitored for Molecular residual disease (MRD) monitoring. lpWGS yielded negative results, targeted NGS sequencing was conducted. Results: Among the enrolled patients (N=34), 21 patients (61.8%) had nodal metastasis and 22 patients (64.7%) were hormone receptor-positive. A total 26 patients (76.5%) achieved pCR. Positive ctDNA was detected in 20 patients (58.8%) at baseline. Higher nodal stage was significantly associated with ctDNA positivity at baseline. After surgery, MRD positivity was observed in 3 patients (8.8%). Among the 26 patents who acheived pCR, only 1 patient (3.8%) showed MRD positivity, while 2 non-pCR patients (25%) demonstrated MRD positivity. Conclusion: The presence of MRD was significantly associated with non-pCR status. The presence of ctDNA at baseline was associated with a higher nodal stage, suggesting its potential as a prognostic marker. Further studies with larger cohorts are warranted to validate these findings and explore the utility of ctDNA analysis for personalized treatment decisions in HER2-positive breast cancer. Citation Format: Byeongju Kang, Soo Jung Lee, Jeeyeon Lee, In Hee Lee, Jee-Young Park, Ho Yong Park, Tiantian Zheng, Lu Tan, Liam Il-Young Chung, Shidong Jia, Pan Du, Young Kwang Chae, Yee Soo Chae. Clinical significance of Circulating tumor DNA (ctDNA) analysis in predicting pathologic complete remission in HER2-positive Breast Cancer Patients Undergoing Neoadjuvant Chemotherapy [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 PO4-01-04.

Next-Generation Sequencing and Emerging Technologies.

Genetic sequencing technologies are evolving at a rapid pace with major implications for research and clinical practice. In this review, the authors provide an updated overview of next-generation sequencing (NGS) and emerging methodologies. NGS has tremendously improved sequencing output while being more time and cost-efficient in comparison to Sanger sequencing. The authors describe short-read sequencing approaches, such as sequencing by synthesis, ion semiconductor sequencing, and nanoball sequencing. Third-generation long-read sequencing now promises to overcome many of the limitations of short-read sequencing, such as the ability to reliably resolve repeat sequences and large genomic rearrangements. By combining complementary methods with massively parallel DNA sequencing, a greater insight into the biological context of disease mechanisms is now possible. Emerging methodologies, such as advances in nanopore technology, in situ nucleic acid sequencing, and microscopy-based sequencing, will continue the rapid evolution of this area. These new technologies hold many potential applications for hematological disorders, with the promise of precision and personalized medical care in the future.

Molecular surveillance of carbapenem-resistant Enterobacterales in two nearby tertiary hospitals to identify regional spread of high-risk clones in Germany, 2019-2020

Understanding the transmission dynamics of carbapenem-resistant Enterobacterales (CRE) is critical to addressing the escalating global threat of antimicrobial resistance (AMR). Although hospital transmission of CRE has been extensively studied, information on community transmission is lacking. This study aims to identify genomic clusters of CRE from two nearby institutions that may be indicative of community or inter-facility transmission. CRE isolates between 1st of January 2019 and 31st of December 2020 from two tertiary hospitals, detected in the respective routine microbiology laboratories, were collected and characterized by short-read whole genome sequencing. A total of 272 CRE were collected, with Enterobacter cloacae complex (71/192, 37%) predominant in Heidelberg and Escherichia coli (19/80, 24%) in Mannheim. The most common carbapenem resistance gene, blaOXA-48, was detected in 38% of CRE from both centres. Several putative transmission clusters were found, including 6 clusters of E. cloacae complex, 5 clusters of Klebsiella pneumoniae, 4 clusters of Citrobacter freundii, and 2 clusters each of Escherichia coli and K. aerogenes. No clusters involved isolates from both study centres, except for an ST22 C. freundii cluster. Globally circulating clones were identified between the two centres for ST131 E. coli, ST66 E. hormaechei and ST22 C. freundii. This study found no widespread transmission clusters among isolates from both centres, suggesting a hospital-specific clonal structure. This suggests that CRE clusters involving both institutions may indicate emerging or circulating clones in the community, highlighting the need for intersectoral surveillance and data sharing.