Library Preparation Strategy Research Articles

Abstract 2937: Accurate detection of human papillomavirus breakpoints in cervical cancers using a novel library preparation strategy for nanopore sequencing technology

Abstract Introduction: Integration of human papillomavirus (HPV) DNA into the human genome is considered as a key event in cervical carcinogenesis. Different methods have been employed in exploring HPV integration sites, such as APOT, DIPS-PCR, and NGS. However, these methods have their drawbacks. APOT heavily relies on the quality of the sample due to its dependence on RNA, while DIPS-PCR mostly identifies known sites and has limited capability in uncovering new integration sites. Although the detection sensitivity of virus infection status increased significantly through the Illumina sequencing platform, there were still disadvantages remain for further improvement, including the detection accuracy and the complex integrated genome structure identification, etc. Also, it requires large amounts of sequencing data, and thus is not applicable in clinical usage, which requires fast and accurate results. Therefore, the development of a new method for HPV integration detection with high accuracy and prompt reporting capacity is crucial for future clinical application. Methods: We established a novel library preparation strategy for the detection of HPV integration in the human genome using Oxford Nanopore sequencing. Using this novel technology, Nanopore libraries were prepared from HPV positive cell lines HeLa and SiHa followed by sequencing on the Oxford Nanopore. We also determined HPV integration sites in five patients with cervical cancer patients with HPV positive tumors using Ilumina WGS. HPV integrations and breakpoints in human genome from both Nanopore and Ilumina WGS were extracted using Minimap and ViFi tool respectively. All HPV integration sites from cell lines and patient samples were validated using Sanger sequencing. Results: The novel library preparation approach for detection of HPV integrations with Nanopore sequencing technology were sensitive and efficient in detection of commonly reported HPV breakpoints in HPV positive cell lines HeLa and SiHa. Additionally, we applied this method to clinical samples from five patients subjected to Whole Genome Sequencing, consistently yielding concordant results. Conclusion: Our Nanopore based assay for detecting HPV integrations without the need for WGS demonstrated high efficacy. These data highlight the sensitivity and efficiency of this methodology in detecting HPV integrations events, emphasizing its potential as a reliable diagnostic tool in understanding pathophysiology of HPV-related malignancies and its use as prognostic markers in clinical settings. Citation Format: Preetiparna Parida, Nivedita Mukherjee, Agastya Singh, Ashima Singh, Krishna Sharan, Mahadev Rao, Shirley Lewis, Sabarinathan Radhakrishnan, Rama Rao Damerla. Accurate detection of human papillomavirus breakpoints in cervical cancers using a novel library preparation strategy for nanopore sequencing technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2937.

A review on the impact of single-stranded library preparation on plasma cell-free diversity for cancer detection.

In clinical oncology, cell-free DNA (cfDNA) has shown immense potential in its ability to noninvasively detect cancer at various stages and monitor the progression of therapy. Despite the rapid improvements in cfDNA liquid biopsy approaches, achieving the required sensitivity to detect rare tumor-derived cfDNA still remains a challenge. For next-generation sequencing, the perceived presentation of cfDNA is strongly linked to the extraction and library preparation protocols. Conventional double-stranded DNA library preparation (dsDNA-LP) focuses on assessing ~167bp double-stranded mononucleosomal (mncfDNA) and its other oligonucleosomal cell-free DNA counterparts in plasma. However, dsDNA-LP methods fail to include short, single-stranded, or nicked DNA in the final library preparation, biasing the representation of the actual cfDNA populations in plasma. The emergence of single-stranded library preparation (ssDNA-LP) strategies over the past decade has now allowed these other populations of cfDNA to be studied from plasma. With the use of ssDNA-LP, single-stranded, nicked, and ultrashort cfDNA can be comprehensively assessed for its molecular characteristics and clinical potential. In this review, we overview the current literature on applications of ssDNA-LP on plasma cfDNA from a potential cancer liquid biopsy perspective. To this end, we discuss the molecular principles of single-stranded DNA adapter ligation, how library preparation contributes to the understanding of native cfDNA characteristics, and the potential for ssDNA-LP to improve the sensitivity of circulating tumor DNA detection. Additionally, we review the current literature on the newly reported species of plasma ultrashort single-stranded cell-free DNA plasma, which appear biologically distinct from mncfDNA. We conclude with a discussion of future perspectives of ssDNA-LP for liquid biopsy endeavors.

Distinct Genetic and Fragmentomic Signatures of Plasma Ctdna Synergize and Resolve Clinical Challenges in Patients with Large B-Cell Lymphoma

Introduction Seizing the full potential of lymphoma circulating tumor DNA (ctDNA) for scientific discoveries and accurate precision medicine tools requires diminished dependence on tumor tissues, enhanced detection of rare variants and harnessing non-random ctDNA fragmentation patterns for non-invasive profiling. Establishment of such tools is pivotal to overcome clinical challenges set by pervasive heterogeneity and different treatment responses in the patients with large B-cell lymphoma (LBCL). Methods Using our previous data on targeted and whole-genome sequencing (WGS) levels of lymphoma ctDNA (Meriranta, 2022), we constructed a novel targeted sequencing assay with B-cell lymphoma-relevant loci and used duplex barcoding to improve analytical sensitivity. We optimized fragment pattern conservation by comparison of different library preparation strategies and established the requirements for exhaustive and reproducible duplex sequencing with error-rate down to 2-4/10^6. We examined replicate samples and in vitro and in silico dilution series of ctDNA spike-in in healthy donor cell-free DNA (cfDNA) to determine the limits of tumor-naïve variant calling, ctDNA quantification and minimal residual disease (MRD) detection. We applied this assay to >500 plasma, tumor and whole-blood DNA samples from primary LBCL patients treated in biomarker driven Nordic phase II trial (Leppä, 2022) and correlated the results with biological and clinical data (Fig A). Results Ultra-sensitive charting of pretreatment ctDNA revealed profound spatial heterogeneity in LBCL patients at diagnosis. Plasma profiles were formed by contributions from multiple cell subpopulations with divergent hypermutations and driver events, some exhibiting convergent trajectories diverged even at early B-cell stages preceding germinal center entrance. The cell populations represented in the diagnostic biopsies contributed an unpredictable proportion of the overall ctDNA pool and majority (63%) of the subclonal ctDNA mutations were not detected in the matched tissues resulting in conflicting genotypes between different materials. Dominant but not subclonal TP53 mutations in the ctDNA associated with poor survival also uncovering patients with no TP53 aberrations in their diagnostic biopsies. On therapy, allele fractions of lymphoma mutations, regardless of their clonal status, declined with early responding patients being MRD negative after two treatment cycles with excellent survival. At the end-of-therapy, Duplex-MRD predicted progression with excellent specificity (98%) but with limited negative predictive value (90%) and sensitivity (47%). Fragmentomic analysis revealed that duplex-recovery was suboptimal for subnucleosomal cfDNA fragment lengths with the highest mutational content. Remarkably, harnessing cfDNA fragments with only single-strand recovery (i.e. non-duplex) for combined ‘Dual-MRD’ enhanced ctDNA detection, translated to better distinction of early responders and resolved most false-positive end-of-therapy FDG-PET scans (Fig A). Besides enriched for shorter fragment sizes, additional non-random structural features of ctDNA molecules extended to their distinct repertoire of fragment nucleotide end motifs (Fig B). Notably, mutant fragments carried less physiological DNAse1L3 nuclease end signatures than their reference counterparts or healthy donor cfDNA (Fig B). Distinct fragmentomic elements of ctDNA molecules contained unexploited utility for non-invasive disease phenotyping and detection that we spanned to shallow (~10x) WGS breadth in independent validation series of lymphoma and healthy donor samples. Conclusion By harnessing synergy between duplex technology, single-strand recovery and distinct fragmentomic features, we discovered novel characteristics in lymphoma ctDNA that correlate with disease biology and improve non-invasive disease profiling to overcome limitations of tumor biopsies and clinical estimates.

A comparison of Oxford nanopore library strategies for bacterial genomics

BackgroundOxford nanopore Technologies (ONT) provides three main library preparation strategies to sequence bacterial genomes. These include tagmentation (TAG), ligation (LIG) and amplification (PCR). Despite ONT’s recommendations, making an informed decision for preparation choice remains difficult without a side-by-side comparison. Here, we sequenced 12 bacterial strains to examine the overall output of these strategies, including sequencing noise, barcoding efficiency and assembly quality based on mapping to curated genomes established herein.ResultsAverage read length ranged closely for TAG and LIG (> 5,000 bp), while being drastically smaller for PCR (< 1,100 bp). LIG produced the largest output with 33.62 Gbp vs. 11.72 Gbp for TAG and 4.79 Gbp for PCR. PCR produced the most sequencing noise with only 22.7% of reads mappable to the curated genomes, vs. 92.9% for LIG and 87.3% for TAG. Output per channel was most homogenous in LIG and most variable in PCR, while intermediate in TAG. Artifactual tandem content was most abundant in PCR (22.5%) and least in LIG and TAG (0.9% and 2.2%). Basecalling and demultiplexing of barcoded libraries resulted in ~ 20% data loss as unclassified reads and 1.5% read leakage.ConclusionThe output of LIG was best (low noise, high read numbers of long lengths), intermediate in TAG (some noise, moderate read numbers of long lengths) and less desirable in PCR (high noise, high read numbers of short lengths). Overall, users should not accept assembly results at face value without careful replicon verification, including the detection of plasmids assembled from leaked reads.

Efficient and sensitive profiling of RNA-protein interactions using TLC-CLIP.

RNA-binding proteins are instrumental for post-transcriptional gene regulation, controlling all aspects throughout the lifecycle of RNA molecules. However, transcriptome-wide methods to profile RNA-protein interactions in vivo remain technically challenging and require large amounts of starting material. Herein, we present an improved library preparation strategy for crosslinking and immunoprecipitation (CLIP) that is based on tailing and ligation of cDNA molecules (TLC). TLC involves the generation of solid-phase cDNA, followed by ribotailing to significantly enhance the efficiency of subsequent adapter ligation. These modifications result in a streamlined, fully bead-based library preparation strategy, which eliminates time-consuming purification procedures and drastically reduces sample loss. As a result, TLC-CLIP displays unparalleled sensitivity, enabling the profiling of RNA-protein interactions from as few as 1000 cells. To demonstrate the effectiveness of TLC-CLIP, we profiled four endogenous RNA-binding proteins, showcasing its reproducibility and improved precision resulting from a higher occurrence of crosslinking-induced deletions. These deletions serve as an intrinsic quality metric and increase both specificity and nucleotide-resolution.

The performance of whole genome bisulfite sequencing on DNBSEQ-Tx platform examined by different library preparation strategies

BackgroundWhole-genome bisulfite sequencing (WGBS) technology can provide comprehensive DNA methylation at a single-base resolution on a genome-wide scale, and is considered to be the gold standard for the detection of 5-methylcytosine (5 mC). However, the International Human Epigenome Consortium propose a full DNA methylome should have at least 30 fold redundant coverage of the reference genome from a single biological replicate. Therefore, it remains cost prohibitive for large-scale studies. To find a solution, the DNBSEQ-Tx sequencing was developed that can generate up to 6 Tb data in a single run for projects involving large-scale sequencing. ResultsIn this study, we provided two WGBS library construction methods DNB_PREBSseq and DNB_SPLATseq optimized for the DNBSEQ-Tx sequencer, and demonstrated the performance of these two methods on the DNBSEQ-Tx platform, using the DNA extracted from four different cell lines. We also compared the sequencing data from these two WGBS library construction methods with HeLa cell line data from ENCODE sequenced on Illumina HiSeq X Ten and WGBS data of two other cell lines sequenced on HiSeq2500. Various quality control (QC) analyses such as the base quality scores, methylation-bias (m-bias), and conversion efficiency indicated that the data sequenced on the DNBSEQ-Tx platform met the WGBS-required quality controls. Meanwhile, our data closely resembled the coverage shown by the data generated by the Illumina platform. ConclusionsOur study showed that with our optimized methods, DNBSEQ-Tx could generate high-quality WGBS data with relatively good stability for large-scale WGBS sequencing applications. Thus, we conclude that DNBSEQ-Tx can be used for a wide range of WGBS research.

Blocking Abundant RNA Transcripts by High-Affinity Oligonucleotides during Transcriptome Library Preparation

BackgroundRNA sequencing has become the gold standard for transcriptome analysis but has an inherent limitation of challenging quantification of low-abundant transcripts. In contrast to microarray technology, RNA sequencing reads are proportionally divided in function of transcript abundance. Therefore, low-abundant RNAs compete against highly abundant - and sometimes non-informative - RNA species.ResultsWe developed an easy-to-use strategy based on high-affinity RNA-binding oligonucleotides to block reverse transcription and PCR amplification of specific RNA transcripts, thereby substantially reducing their abundance in the final sequencing library. To demonstrate the broad application potential of our method, we applied it to different transcripts and library preparation strategies, including YRNAs in small RNA sequencing of human blood plasma, mitochondrial rRNAs in both 3′ end sequencing and long-read sequencing, and MALAT1 in single-cell 3′ end sequencing. We demonstrate that the blocking strategy is highly efficient, reproducible, specific, and generally results in better transcriptome coverage and complexity.ConclusionOur method does not require modifications of the library preparation procedure apart from simply adding blocking oligonucleotides to the RT reaction and can thus be easily integrated into virtually any RNA sequencing library preparation protocol.

Abstract 5791: Microfluidics-based library preparation strategies for targeted high-throughput sequencing and RNA sequencing in hematological malignancies in clinical samples

Abstract Introduction We developed a unique panel of 54 target genes dedicated to hematological malignancies (HematoPanel) and screened 1,108 clinical samples. We also performed transcriptomic analysis and evaluated its relevance for patients with acute lymphoblastic leukemia (ALL). Methods DNA and RNA were extracted from cell pellets with the Maxwell® RSC (Promega®). Libraries for targeted high-throughput DNA sequencing (HTS) were generated in duplicate from 100 ng with the Advanta™ NGS Library Prep Assay kit using the LP 48.48 IFC (integrated fluidic circuit) on a Juno™ and sequenced on the NextSeq™ 550 (Illumina®) to a median depth of coverage of approximately 1,200X per sample. Transcriptomic libraries were generated from 50 ng RNA with the Advanta RNA-Seq XT NGS Library Prep Kit using the LP 48.Atlas™ IFC on Juno and sequenced on the NextSeq 550. A bioinformatics pipeline was developed for fusion transcript detection. Differential gene expression was analyzed with the DESeq2 package after STAR alignment. Results In 2020 we sequenced 1,108 consecutive samples in routine practice with HematoPanel. The main indications were the suspicion of myeloproliferative neoplasms (32%), the evaluation of chronic lymphocytic leukemia (23%), and the diagnostic workup of cytopenias (15%). We detected 1,306 mutations in 654 patients (59%) (1 to 8 mutations per sample). Eight genes (TET2, SF3B1, TP53, DNMT3A, ASXL1, JAK2, SRSF2 and MYD88) were reported as mutated for more than 50 patients. We sequenced 22 samples including the MOLT-4 cell line, healthy donor samples, and 16 ALL samples for transcriptomic evaluation. From ALL samples, we detected 5 fusion transcripts previously identified by specific PCR (1 BCR-ABL, 2 ETV6-RUNX1, 1 KMT2A-AFF1, 1 TCF3-PBX1). Moreover, we detected 4 other fusion transcripts for 3 patients (EBF1-PDGFRB, FGFR1-MYO18A, P2RY8-CRLF2, and PAX5-NOL4L). Interestingly, these patients clustered with the BCR-ABL positive sample based on their expression profile. It confirms the diagnosis of Phi-like ALL and thus offers a therapeutic target for these patients. Conclusion Microfluidics-based library prep using Juno offers a cost-effective strategy and straightforward workflows for both targeted HTS and RNA sequencing. We validated its use for hematological malignancy evaluation in routine practice. Citation Format: Cédric Pastoret, Amyra Aliouat, Marie-Laure Boulland, Florent Denoual, Anne Desmares, Marie De Tayrac, Thierry Fest, Celeste Letellier. Microfluidics-based library preparation strategies for targeted high-throughput sequencing and RNA sequencing in hematological malignancies in clinical samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5791.

Riboviz 2: a flexible and robust ribosome profiling data analysis and visualization workflow.

MotivationRibosome profiling, or Ribo-seq, is the state-of-the-art method for quantifying protein synthesis in living cells. Computational analysis of Ribo-seq data remains challenging due to the complexity of the procedure, as well as variations introduced for specific organisms or specialized analyses.ResultsWe present riboviz 2, an updated riboviz package, for the comprehensive transcript-centric analysis and visualization of Ribo-seq data. riboviz 2 includes an analysis workflow built on the Nextflow workflow management system for end-to-end processing of Ribo-seq data. riboviz 2 has been extensively tested on diverse species and library preparation strategies, including multiplexed samples. riboviz 2 is flexible and uses open, documented file formats, allowing users to integrate new analyses with the pipeline.Availability and implementationriboviz 2 is freely available at github.com/riboviz/riboviz.

Plasma Cell Disease Genotyping with the Liquid Biopsy

IntroductionEasily accessible, real-time genotyping is desirable for patients suffering from plasma cell (PC) disorders for diagnostic, prognostic and therapeutic purpose. However, PC disorders usually lack a leukemic phase, and up to now genotyping of tumor cells required purified material from bone marrow (BM) or tissue biopsies. Circulating cell-free DNA (cfDNA) might be an accessible source of tumor material in patients with PC diseases to identify cancer-gene somatic mutations. Accessing the peripheral blood (PB) has clear advantages with respect to the sampling procedure itself, and has the potential to better reflect tumor heterogeneity. Here we aimed at tracking the genetic profile of different PC dyscrasias using plasma cfDNA.MethodsTwenty-eight consecutive patients (median age= 64; male:female ratio=15:13) with PC disorders [two with monoclonal gammopathy of undetermined significance (MGUS), five with smoldering MM (SMM), and 21 with symptomatic MM] were included between September 2016 and May 2017. The following material was collected: (1) cfDNA isolated from plasma, (2) tumor genomic DNA (gDNA) from CD138+ purified BM PCs for comparative purposes, and (3) normal germ line gDNA extracted from PB granulocytes after Ficoll separation, to filter out polymorphisms. The sampling was done at diagnosis in 25 patients, and during the course of the disease in three MM cases. Median BM PC infiltration was 40% (range: 7-90%). A targeted resequencing gene panel, including coding exons and splice sites of 14 genes (target region: 31 kb) was specifically designed and optimized to allow a priori the recovery of at least one clonal mutation in 68% (95% confidence interval: 58-76) of MM patients. Ultra-deep next-generation sequencing (NGS) of the gene panel was performed on MiSeq (Illumina) using the CAPP-seq library preparation strategy (NimbleGen). The somatic function of VarScan2 was used to call non-synonymous somatic mutations, and a stringent bioinformatic pipeline was developed and applied to filter out sequencing errors (detection limit 3x10-3). The sensitivity and specificity of plasma cfDNA genotyping were calculated in comparison with tumor gDNA genotyping as the gold standard.ResultsIn this prospectively collected cohort of 28 unselected PC dyscrasia patients, circulating tumor cfDNA was detectable in plasma samples with a median of 12011.60 haploid genome-equivalents per mL. The application of our targeted ultra-deep NGS approach for plasma cfDNA genotyping resulted in ≥90% of the target region covered ˃1000X in all plasma samples, and ≥90% of the target region covered ˃2000X in 23/28. Overall, within the interrogated genes, 18/28 (64%) patients harbored somatic mutations (total number: 28; range: 1-4 mutations per patient) that were detectable in plasma cfDNA. Quite consistent with the typical spectrum of mutated genes in MM, plasma cfDNA genotyping revealed somatic variants of NRAS in 25%; KRAS in 14%; TP53, TRAF3 and FAM46C in 11%, respectively; CYLD and DIS3 in 7%, respectively; and BRAF and IRF4 in 4% of cases, respectively (Figure 1A). By comparing the genotype of circulating tumor cfDNA with that of gDNA from purified malignant BM PCs of the diagnostic biopsy (gold standard) (Figure 1B), we found that cfDNA genotyping correctly identified 72% of the mutations confirmed in the tumor biopsy (Figure 1C). Notably, the remaining mutations not discovered in cfDNA had a low representation in the purified BM PCs (median allelic abundance=2.5%; range 1.1-4.96%). ROC analysis showed that circulating tumor cfDNA genotyping had the highest sensitivity (92.9%) if mutations were represented in >5% of the alleles of the purified BM PCs (Figures 1D-E). In none of the cases, cfDNA genotyping identified additional somatic mutations not detected in the purified BM PCs.ConclusionsOverall, our results provide the proof of principle that circulating tumor cfDNA genotyping is a feasible, non-invasive real-time approach that reliably detects clonal and subclonal somatic mutations represented in at least 5% of alleles in tumor PCs. [Display omitted] DisclosuresBertoni:Acerta Pharma: Research Funding; Bayer: Research Funding; Cellestia: Research Funding; Menarini: Research Funding; Piqur: Research Funding; Immunogen: Research Funding. Zucca:Celgene: Honoraria, Research Funding; Jannsen: Consultancy, Honoraria, Other: Advisory role; Mundipharma: Research Funding; Celgene: Honoraria, Research Funding; Roche: Advisory role, Honoraria, Research Funding; Roche: Honoraria, Research Funding; Mundipharma: Research Funding; Gilead Science: Consultancy, Other: Advisory role; Sandoz: Consultancy, Other: Advisory role; Jannsen: Consultancy, Honoraria, Other: Advisory role; Gilead Science: Consultancy, Other: Advisory role; Celltrion Healthcare: Consultancy, Other: Advisory Role; Bayer: Consultancy, Other: Advisory Role; Sandoz: Consultancy, Other: Advisory role; Takeda: Consultancy, Other: Advisory role; Celltrion Healthcare: Consultancy, Other: Advisory Role; Takeda: Consultancy, Other: Advisory role; Bayer: Consultancy, Other: Advisory Role.

Genotyping of Classical Hodgkin Lymphoma on the Liquid Biopsy

INTRODUCTION. In classical Hodgkin lymphoma (cHL), the low representation (~5%) of Hodgkin-Reed-Sternberg cells (HRS) challenged tumor genotyping on the tissue biopsy. Cell free DNA (cfDNA) is shed into the blood by tumor cells and can be used as source of tumor DNA for the identification of somatic mutations, track clonal evolution of tumors and detect minimal residual disease during therapy. AIMS. The study aimed at: i) showing that cfDNA mirrors the genetics of HRS cells in cHL patients; ii) characterizing the mutational profile of a large cohort of newly diagnosed and chemorefractory cHL; iii) identifying molecular prognostic subtypes; iv) early detecting residual disease during therapy; and v) longitudinally tracking tumor clonal evolution under different treatment modalities. METHODS. The study included 80 newly diagnosed cHL and 32 chemorefractory cHL. The following biological material was analyzed: i) cfDNA from plasma collected at diagnosis, during ABVD courses, at refractory progression, before and during therapy with brentuximab or nivolumab; and ii) normal germline genomic DNA (gDNA) from granulocytes. For comparative purposes, paired tumor gDNA from microdissected HRS cells of 13 cases was also analyzed. A targeted resequencing panel optimized to include the coding exons and splice sites of 77 genes recurrently mutated in B-cell lymphomas was used for genotyping. Ultra-deep next-generation sequencing (NGS) of the gene panel was performed on NexSeq 500 (Illumina) using the CAPP-seq library preparation strategy (NimbleGen). RESULTS. In cHL patients, cfDNA surrogated gDNA from HRS cells, since it harbored 87.5% of the tumor confirmed mutations. Genes recurrently affected by non-synonymous somatic mutations in >20% of cHL included STAT6 (37.5%), TNFAIP3 (35%), and ITPKB (27.5%) (Fig. 1A). Mutations clustered in major pathways, including NF-κB, PI3K-AKT, cytokine and NOTCH signaling, and immune evasion. ITPKB mutations: i) were quite specific for cHL, being rare or absent in other lymphomas; ii) caused the subcellular delocalization of the protein in primary HRS cells of mutated patients; iii) correlated with clues of PI3K-AKT signaling activation both at gene expression and protein levels; and iv) consistent with the positioning of ITPKB downstream PI3K in the pathway, associate with resistance to PI3K inhibitors. Mutations of CD58, encoding a co-stimulatory molecule for T-cells, associated with short PFS independent of interim PET/CT results, pointing to immune escape genetic lesions as biomarkers of aggressive disease (Fig. 1B). Newly diagnosed and chemorefractory cHL shared a largely overlapping mutational landscape. TP53 mutations were not enriched in refractory cHL as instead commonly found in other types of refractory B-cell tumors. Conversely, more TET2 mutations were documented in refractory cHL, including newly acquired mutation, thus signaling towards aberrant DNA methylation programming as a mechanism of resistance in cHL with potential therapeutic implications. By longitudinal analysis, in patients relapsing under/after chemotherapy or brentuximab vedotin, pre-treatment/relapse tumor pairs branched through the acquisition of phase specific mutations from an ancestral clone, that always persisted (Fig. 1C). Conversely, in patients maintained under nivolumab, clones were cyclically suppressed and replaced by completely novel clones. We utilized the change in circulating tumor cfDNA load from baseline to interim timepoint to predict the best response to ABVD and to complement interim PET/CT in anticipating cure. A drop of 100-fold or 2-log drop in tumor cfDNA after 2 ABVD courses associated with an eventual complete response and cure. All cured patients that were inconsistently judged as interim PET/CT positive turned out to have a >2 log drop in tumor cfDNA. A drop of less than 2-log in tumor cfDNA after 2 ABVD courses associated with an eventual progression. All relapsed patients that were inconsistently judged as interim PET/CT negative turned out to have a <2 log drop in tumor cfDNA (Fig. 1D). CONCLUSIONS. Circulating tumor cfDNA allows to noninvasively detect tumor-specific mutations: i) identify prognostic subtypes; ii) early detect residual disease during therapy; and iii) longitudinally track tumor clonal evolution under different treatment modalities. [Display omitted] DisclosuresStathis:Celgene: Research Funding; Pfizer: Research Funding; Merck: Research Funding; Roche: Consultancy, Other: Advisory board; Amgen: Honoraria. Bertoni:Acerta Pharma: Research Funding; Bayer: Research Funding; Cellestia: Research Funding; Menarini: Research Funding; Piqur: Research Funding; Immunogen: Research Funding. Santoro:Merck: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Zucca:Bayer: Consultancy, Other: Advisory Role; Jannsen: Consultancy, Honoraria, Other: Advisory role; Takeda: Consultancy, Other: Advisory role; Celltrion Healthcare: Consultancy, Other: Advisory Role; Celltrion Healthcare: Consultancy, Other: Advisory Role; Celgene: Honoraria, Research Funding; Sandoz: Consultancy, Other: Advisory role; Bayer: Consultancy, Other: Advisory Role; Gilead Science: Consultancy, Other: Advisory role; Roche: Honoraria, Research Funding; Jannsen: Consultancy, Honoraria, Other: Advisory role; Roche: Advisory role, Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Mundipharma: Research Funding; Mundipharma: Research Funding; Gilead Science: Consultancy, Other: Advisory role; Takeda: Consultancy, Other: Advisory role; Sandoz: Consultancy, Other: Advisory role. Gaidano:Roche: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Amgen: Consultancy, Honoraria. Carlo-Stella:ADC Therapeutics: Research Funding.

In Vivo Generation of BK and JC Polyomavirus Defective Viral Genomes in Human Urine Samples Associated with Higher Viral Loads.

ABSTRACTDefective viral genomes (DVGs) are parasitic viral sequences containing point mutations, deletions, or duplications that might interfere with replication. DVGs are often associated with viral passage at high multiplicities of infection in culture systems but have been increasingly reported in clinical specimens. To date however, only RNA viruses have been shown to contain DVGs in clinical specimens. Here, using direct deep sequencing with multiple library preparation strategies and confirmatory digital droplet PCR (ddPCR) of urine samples taken from immunosuppressed individuals, we show that clinical BK polyomavirus (BKPyV) and JC polyomavirus (JCPyV) strains contain widespread genomic rearrangements across multiple loci that likely interfere with viral replication. BKPyV DVGs were derived from BKPyV genotypes Ia, Ib-1, and Ic. The presence of DVGs was associated with specimens containing higher viral loads but never reached clonality, consistent with a model of parasitized replication. These DVGs persisted during clinical infection as evidenced in two separate pairs of samples containing BK virus collected from the same individual up to 302 days apart. In a separate individual, we observed the generation of DVGs after a 57.5-fold increase in viral load. In summary, by extending the presence of DVGs in clinical specimens to DNA viruses, we demonstrate the ubiquity of DVGs in clinical virology.IMPORTANCE Defective viral genomes (DVGs) can have a significant impact on the production of infectious virus particles. DVGs have only been identified in cultured viruses passaged at high multiplicities of infection and RNA viruses collected from clinical specimens; no DNA virus in the wild has been shown to contain DVGs. Here, we identified BK and JC polyomavirus DVGs in clinical urine specimens and demonstrated that these DVGs are more frequently identified in samples with higher viral loads. The strains containing DVGs had rearrangements throughout their genomes, with the majority affecting genes required for viral replication. Longitudinal analysis showed that these DVGs can persist during an infection but do not reach clonality within the chronically infected host. Our identification of polyomavirus DVGs suggests that these parasitic sequences exist across the many classes of viruses capable of causing human disease.

Chromatin conformation capture (Hi-C) sequencing of patient-derived xenografts: analysis guidelines.

Sequencing of patient-derived xenograft (PDX) mouse models allows investigation of the molecular mechanisms of human tumor samples engrafted in a mouse host. Thus, both human and mouse genetic material is sequenced. Several methods have been developed to remove mouse sequencing reads from RNA-seq or exome sequencing PDX data and improve the downstream signal. However, for more recent chromatin conformation capture technologies (Hi-C), the effect of mouse reads remains undefined. We evaluated the effect of mouse read removal on the quality of Hi-C data using in silico created PDX Hi-C data with 10% and 30% mouse reads. Additionally, we generated 2 experimental PDX Hi-C datasets using different library preparation strategies. We evaluated 3 alignment strategies (Direct, Xenome, Combined) and 3 pipelines (Juicer, HiC-Pro, HiCExplorer) on Hi-C data quality. Removal of mouse reads had little-to-no effect on data quality as compared with the results obtained with the Direct alignment strategy. Juicer extracted more valid chromatin interactions for Hi-C matrices, regardless of the mouse read removal strategy. However, the pipeline effect was minimal, while the library preparation strategy had the largest effect on all quality metrics. Together, our study presents comprehensive guidelines on PDX Hi-C data processing.

Mutational profiling of micro-dissected pre-malignant lesions from archived specimens

BackgroundSystematic cancer screening has led to the increased detection of pre-malignant lesions (PMLs). The absence of reliable prognostic markers has led mostly to over treatment resulting in potentially unnecessary stress, or insufficient treatment and avoidable progression. Importantly, most mutational profiling studies have relied on PML synchronous to invasive cancer, or performed in patients without outcome information, hence limiting their utility for biomarker discovery. The limitations in comprehensive mutational profiling of PMLs are in large part due to the significant technical and methodological challenges: most PML specimens are small, fixed in formalin and paraffin embedded (FFPE) and lack matching normal DNA.MethodsUsing test DNA from a highly degraded FFPE specimen, multiple targeted sequencing approaches were evaluated, varying DNA input amount (3–200 ng), library preparation strategy (BE: Blunt-End, SS: Single-Strand, AT: A-Tailing) and target size (whole exome vs. cancer gene panel). Variants in high-input DNA from FFPE and mirrored frozen specimens were used for PML-specific variant calling training and testing, respectively. The resulting approach was applied to profile and compare multiple regions micro-dissected (mean area 5 mm2) from 3 breast ductal carcinoma in situ (DCIS).ResultsUsing low-input FFPE DNA, BE and SS libraries resulted in 4.9 and 3.7 increase over AT libraries in the fraction of whole exome covered at 20x (BE:87%, SS:63%, AT:17%). Compared to high-confidence somatic mutations from frozen specimens, PML-specific variant filtering increased recall (BE:85%, SS:80%, AT:75%) and precision (BE:93%, SS:91%, AT:84%) to levels expected from sampling variation. Copy number alterations were consistent across all tested approaches and only impacted by the design of the capture probe-set. Applied to DNA extracted from 9 micro-dissected regions (8 PML, 1 normal epithelium), the approach achieved comparable performance, illustrated the data adequacy to identify candidate driver events (GATA3 mutations, ERBB2 or FGFR1 gains, TP53 loss) and measure intra-lesion genetic heterogeneity.ConclusionAlternate experimental and analytical strategies increased the accuracy of DNA sequencing from archived micro-dissected PML regions, supporting the deeper molecular characterization of early cancer lesions and achieving a critical milestone in the development of biology-informed prognostic markers and precision chemo-prevention strategies.

Damage patterns observed in mtDNA control region MPS data for a range of template concentrations and when using different amplification approaches.

Massively parallel sequencing (MPS) of mitochondrial (mt) DNA allows practitioners the ability to fully resolve heteroplasmic sites. In forensic DNA analysis, identifying heteroplasmy (a naturally occurring mixture of two mtDNA profiles) can provide additional mtDNA profile information which can lead to an increase in the discrimination potential of an mtDNA match between an evidentiary sample and reference source. Forensic samples such as hair and skeletal remains, especially older, more compromised samples, can often exhibit DNA damage. Because both damage and heteroplasmy can manifest as a mixture of two nucleotides, it is important to differentiate between the two conditions when interpreting mtDNA MPS data. In this study, DNA damage was applied under controlled conditions to samples containing a range of template concentrations, including some with identified heteroplasmy. Damage was applied via storage in water at room temperature on samples diluted before or after storage to mimic low template scenarios. Damage was assessed with respect to the following areas: mtDNA quantification and degradation ratios, MPS read depth, MPS profile results, overall damage rates, and the interpretation of heteroplasmy. Datasets were generated to assess and compare two different amplification and library preparation strategies: the Promega PowerSeq™ CRM Nested System kit and a 1.16kb target amplicon of the entire mtDNA control region followed by a Nextera® XT library preparation. The results of this study provide an evaluation of the Promega 10-plex MPS procedure as an improved process to mitigate the impact of mtDNA damage on low template samples. Some of the negative effects of damage observed in this study were a decrease in mtDNA yield by 20-30% and lower quality MPS sequencing results. These effects were observed more frequently when samples were diluted prior to inducing damage, illustrating that low template samples are more susceptible to damage. The findings of this study will assist forensic laboratories in differentiating between damage and heteroplasmy, which is essential when developing robust mtDNA MPS interpretation guidelines such as setting appropriate reporting thresholds.

Abstract 2499: Genomic spatial profiling of archived pre-malignant lesions of the breast epithelium

Abstract The widespread adoption of breast cancer screening has increased the detection of pre-malignant lesions such as ductal carcinoma in situ (DCIS) but there are currently no reliable biomarkers to stratify DCIS and avoid over-treatment. Importantly, the identification of novel biomarkers in DCIS is notoriously difficult. The rare and late recurrence to subsequent breast cancer (10% at 10 years) poses challenges in experimental design that only a few established, retrospective studies can solve. Most tissue specimens in the associated registries are small, fixed and archived in paraffin (FFPE), precluding their characterization via standard genomic assays. Besides, pure DCIS often present multi-focal heterogenous histology of unclear mutational make-up. Here, we present an approach combining laser capture microdissection with state-of-the-art genomic library preparation methods to enable molecular profiling from minute amounts of damaged nucleic acids and demonstrate its validity for the spatial molecular characterization of DCIS specimen. Targeted DNA sequencing was performed on limited dilutions of a test DNA from archived breast tissue. Three library preparation strategies were evaluated, included adaptation of classic A-tailing (AT), alternate single-strand (SS) or blunt-end (BE) adapter ligations. The use of an AT protocol resulted less than 10% of targeted base pairs covered at more than 20x (Cov20) from lower than 50ng of DNA and suggested the adapter ligation as initial bottleneck. In contrast, SS or BE adapter ligation from 3ng of DNA reduced the fraction of duplicate reads, increasing the Cov20 to 49% and 100% respectively. Artifactual C to T substitutions, a signature of formalin damage, were restricted to variants of low allelic fraction (&amp;lt;5%). As a result, the use of ensemble variant calling and heuristic filtering to eliminate artifacts and germline variants, led to exome-wide mutational profile of 89% precision and 88% recall from 3ng input, comparable to replicate deep sequencing from a mirrored fresh frozen specimen. The presence of an ERBB2 amplification was validated in all tested conditions. The BE approach for whole exomes was validated on DCIS regions (N=8) and normal epithelium (N=1) dissected from 3 FFPE DCIS blocks. The normal epithelium was unaltered and copy number alterations both consistent and inconsistent within specimen were identified, including FGFR1 and ERBB2 gains or TP53 loss restricted to high-grade lesions. Short variants and indels were rare (~123 per region), mostly shared within each specimen and unlikely to represent driver mutations. The proposed approach can support the spatial mutational profiling of pre-malignant lesions. Compatible with a limited quantity of tissue from archived specimens, the approach sets a new paradigm for the retrospective molecular characterization of specimen with longitudinal follow-up and associated long-term outcome. Citation Format: Daniela Nachmanson, Joseph Steward, Taeyong Kim, Huazhen Yao, Eliza Jeong, Thomas O'Keefe, Gillian L. Hirst, Laura Esserman, Alexander Borowsky, Farnaz Hasteh, Kristen Jepsen, Olivier Harismendy. Genomic spatial profiling of archived pre-malignant lesions of the breast epithelium [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 2499.

Abstract P4-05-05: A high-sensitivity molecular profiling approach to spatially characterize archived pre-cancer lesions

Abstract Background: The widespread adoption of breast cancer screening has increased the detection of pre-malignant lesions such as ductal carcinoma in situ (DCIS) but has not translated into increased survival rates. There are currently no reliable biomarkers to stratify DCIS into those which would not develop into an aggressive invasive cancer from those that do, and avoid over-treatment. The interaction with the DCIS microenvironment has likely been under-appreciated or not studied in the adequate clinical context. Importantly, the identification of novel biomarkers in DCIS is notoriously difficult. The rare and late recurrence to subsequent breast cancer (10% at 10 years) poses challenges in experimental design that only a few established, retrospective studies can solve. Most tissue specimens in the associated registries are small, fixed and archived in paraffin (FFPE), precluding their characterization via standard genomic assays. Here, we present an approach combining laser capture microdissection (LCM) with state-of-the-art genomic library preparation methods to enable molecular profiling from minute amounts of damaged nucleic acids and demonstrate its validity for the spatial molecular characterization of DCIS specimen. Methods: Targeted DNA sequencing (whole exome and cancer gene panel) was performed on limited dilutions (200, 50, 10,3 ng) of a test DNA from archived breast tissue, or validation DNA from 10 LCM regions from 3 DCIS specimens. Three library preparation strategies were evaluated, included adaptation of classic A-tailing (AT), or alternate single-strand (SS) or blunt-end (BE) adapter ligations. The expression profiling was performed using SMART-3SEQ protocol on 15 LCM regions from 8 DCIS specimen. Results: The use of an FFPE-optimized hybrid-capture AT protocol resulted in poor coverage performance (7% of targeted base pairs covered at ≥20x) from test DNA inputs lower than 50 ng and suggested the ligation of the sequencing adapter as the initial bottleneck. Alternatively steps using SS or BE adapter ligation from 3 ng of test DNA (~500 cells) increased the library complexity, reducing the fraction of duplicate reads and increasing the fraction of targeted base pairs covered at 20x to 49% and 100% respectively. Artifactual cytosine to tyrosine substitutions, a signature of formalin damage, were restricted to variants of low allelic fraction (&amp;lt;5%). As a result, the use of molecular barcodes, ensemble variant calling and heuristic variant filtering, resulted in 98% precision and 96% recall in variant calling accuracy from 3ng input, when compared to 200 ng input from a fresh frozen mirrored specimen. Similarly, the presence of an ERBB2 amplification was validated in all tested conditions. The validation of the BE approach on 10 LCM regions from 3 DCIS showed decreasing levels of clonal relationships when comparing regions from adjacent sections, then from matching distinct regions, then from distinct specimen. The heterogeneity of TP53 loss of heterozygosity within a specimen was consistent with high-grade. Furthermore, gene expression profiling of 15 regions from 8 archived DCIS specimen differentiated expression profiles of stromal and epithelial regions, within and between specimen, highlighting sets of genes associated with DCIS subtype and the immune make-up of the micro-environment. Conclusion: The proposed approach - from coordinated specimen preparation to mutational and expression profiling - was therefore validated to support the spatial characterization of pre-cancerous lesions and the microenvironment in which they evolve. Compatible with a limited quantity of tissue from archived specimens, the approach sets a new paradigm for the retrospective molecular characterization of specimen with longitudinal follow-up and associated long-term outcome. Citation Format: Daniela Nachamanson, Joseph Steward, Taeyong Kim, Huazhen Yao, Eliza Jeong, Thomas O'Keefe, Gillian L. Hirst, Farnaz Hasteh, Kristen Jepsen, Laura J. Esserman, Alexander Borowsky, Olivier Harismendy. A high-sensitivity molecular profiling approach to spatially characterize archived pre-cancer lesions [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-05-05.

Practical stability of whole-genome bisulfite sequencing using plasma cell-free DNA

With the development of liquid biopsy technology, plasma cell-free DNA (cfDNA) becomes one of the research hotspots. Whole-genome bisulfite sequencing of plasma cell-free DNA has shown great potential medical applications such as cancer detection. However, the practical stability evaluation is still lacking. In this study, plasma cell-free DNA samples from two volunteers at different time were collected and prepared for sequencing in multiple laboratories. The library preparation strategies include pre-bisulfite, post-bisulfite and regular whole-genome sequencing. We established a set of quality control references for plasma cell-free DNA sequencing data and evaluated practical stability of blood collection, DNA extraction, and library preparation and sequencing depth. This work provided a technical practice guide for the application of plasma cfDNA methylation sequencing for clinical applications.

Development of a RACE-based RNA-Seq approach to characterize the T-cell receptor repertoire of porcine γδ T cells

Recent data suggest that porcine γδ T cells exhibit a similar degree of functional plasticity as human and murine γδ T cells. Due to the high frequency of TCR-γδ+ cells in blood and secondary lymphatic organs, the pig is an attractive model to study these cells, especially their combined features of the innate and the adaptive immune system. Using a 5’ RACE-like approach, we translated a human/murine NGS library preparation strategy to capture full-length V-(D)-J TRG and TRD clonotypes in swine. After oligo(dT) primed conversion of input RNA, the cDNA population was enriched for full-length V(D)J TCR transcripts with porcine-specific primers including Illumina adaptor sequences as overhangs for Illumina MiSeq analysis. After quality control and processing by FastQC and ea-utils, porcine TRG and TRD sequences were mapped against the human IMGT reference directory. Porcine blood-derived CD2+ and CD2‾ TCR-γδ+ cells exhibited two distinct clonotypes Vγ11JγP1 (74.6%) and Vγ10JγP1 (57.7%), respectively. Despite the high TCR-δ diversity among CD2+ cells (39 clonotypes), both subsets shared the same abundant Vδ1DδxJδ4 clonotype at approximately identically frequencies (CD2+: 31.2%; CD2‾: 37.0%). The flexible nature of this approach will facilitate the assessment of organ-specific phenotypes of γδ T cell subsets alongside with their respective TCR diversity at single cell resolution.

Adapterama II: universal amplicon sequencing on Illumina platforms (TaggiMatrix).

Next-generation sequencing (NGS) of amplicons is used in a wide variety of contexts. In many cases, NGS amplicon sequencing remains overly expensive and inflexible, with library preparation strategies relying upon the fusion of locus-specific primers to full-length adapter sequences with a single identifying sequence or ligating adapters onto PCR products. In Adapterama I, we presented universal stubs and primers to produce thousands of unique index combinations and a modifiable system for incorporating them into Illumina libraries. Here, we describe multiple ways to use the Adapterama system and other approaches for amplicon sequencing on Illumina instruments. In the variant we use most frequently for large-scale projects, we fuse partial adapter sequences (TruSeq or Nextera) onto the 5′ end of locus-specific PCR primers with variable-length tag sequences between the adapter and locus-specific sequences. These fusion primers can be used combinatorially to amplify samples within a 96-well plate (8 forward primers + 12 reverse primers yield 8 × 12 = 96 combinations), and the resulting amplicons can be pooled. The initial PCR products then serve as template for a second round of PCR with dual-indexed iTru or iNext primers (also used combinatorially) to make full-length libraries. The resulting quadruple-indexed amplicons have diversity at most base positions and can be pooled with any standard Illumina library for sequencing. The number of sequencing reads from the amplicon pools can be adjusted, facilitating deep sequencing when required or reducing sequencing costs per sample to an economically trivial amount when deep coverage is not needed. We demonstrate the utility and versatility of our approaches with results from six projects using different implementations of our protocols. Thus, we show that these methods facilitate amplicon library construction for Illumina instruments at reduced cost with increased flexibility. A simple web page to design fusion primers compatible with iTru primers is available at: http://baddna.uga.edu/tools-taggi.html. A fast and easy to use program to demultiplex amplicon pools with internal indexes is available at: https://github.com/lefeverde/Mr_Demuxy.