Low Input Amounts Research Articles

A Novel Molecular-Genetic Approach to the Monitoring of Dynamics of Mitochondrial Function Improvement during Treatment.

Making a correct genetically based diagnosis in patients with diseases associated with mitochondrial dysfunction can be challenging both genetically and clinically, as can further management of such patients on the basis of molecular-genetic data assessing the state of their mitochondria. In this opinion article, we propose a novel approach (which may result in a clinical protocol) to the use of a precise molecular-genetic tool in order to monitor the state of mitochondria (which reflects their function) during treatment of certain conditions, by means of not only signs and symptoms but also the molecular-genetic basis of the current condition. This is an example of application of personalized genomic medicine at the intersection of a person's mitochondrial genome information and clinical care. Advantages of the proposed approach are its relatively low cost (compared to various types of sequencing), an ability to use samples with a low input amount of genetic material, and rapidness. When this approach receives positive outside reviews and gets an approval of experts in the field (in terms of the standards), it may then be picked up by other developers and introduced into clinical practice.

Abstract 326: Matched fresh frozen and FFPE patient tissues reveal the enhanced sensitivity and data quality of a novel DNA library prep method

Abstract In cancer genomics, a common source of DNA is formalin-fixed, paraffin-embedded (FFPE) tissue from patient surgical samples, where in most cases high quality fresh or frozen tissue samples are not available. FFPE DNA poses many notable challenges for preparing NGS libraries, including low input amounts and highly variable damage from fixation, storage, and extraction methods. It is difficult to obtain libraries with sufficient coverage and the sequencing artifacts arising from damaged DNA bases confound somatic variant detection. Additionally, many laboratories process FFPE tumor samples alongside matched, high quality, normal DNA and many library prep workflows are not readily compatible with both sample types. We developed a novel NGS library prep method compatible with both high quality and very low quality FFPE DNA samples, employing a novel enzymatic DNA repair mix, enzymatic fragmentation mix, and PCR master mix. To validate this workflow on real patient sample sets, we obtained DNA extracted from matched tumor and normal tissue of various tissue types preserved by both fresh frozen and FFPE methods. The FFPE DNA samples ranged in quality from DNA integrity number (DIN) 1.5 to 6.8, including samples stored for nearly 3 decades. We prepared libraries using this method compared against other library prep workflows and sequenced them by WGS and target capture. We assessed the performance of these workflows by library yield, library quality metrics, depth and evenness of coverage, and somatic variant calling with fresh frozen-extracted DNA providing the gold standard for library quality and mutation content. This new enzymatic fragmentation-based library prep workflow not only reduced the false positive rate in somatic variant detection by repairing damage-derived mutations in FFPE DNA samples but also improved the library yield, library quality metrics (including mapping, chimeras, and properly-paired reads), library complexity, coverage depth and uniformity, and hybrid capture library quality metrics. Comparing the variant calls from matched FFPE and frozen tissues revealed an improved sensitivity and accuracy of variant calling using this library prep method compared to mechanical shearing and other enzymatic fragmentation library prep approaches. This new suite of enzyme mixes allows even highly damaged FFPE samples to achieve high quality libraries with a greater sensitivity for somatic variant identification. The workflow is robust and flexible, compatible with both FFPE DNA and matched high quality DNA samples as well as automation-friendly for convenience in sample processing. Citation Format: Margaret R. Heider, Jian Sun, Brittany Sexton, Bradley W. Langhorst, Laura Blum, Pingfang Liu. Matched fresh frozen and FFPE patient tissues reveal the enhanced sensitivity and data quality of a novel DNA library prep method [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 326.

An Automated and Fast Sample Preparation Workflow for Laser Microdissection Guided Ultrasensitive Proteomics

Spatial tissue proteomics integrating whole-slide imaging, laser microdissection, and ultrasensitive mass spectrometry is a powerful approach to link cellular phenotypes to functional proteome states in (patho)physiology. To be applicable to large patient cohorts and low sample input amounts, including single-cell applications, loss-minimized and streamlined end-to-end workflows are key. We here introduce an automated sample preparation protocol for laser microdissected samples utilizing the cellenONE robotic system, which has the capacity to process 192 samples in 3 h. Following laser microdissection collection directly into the proteoCHIP LF 48 or EVO 96 chip, our optimized protocol facilitates lysis, formalin de-crosslinking, and tryptic digest of low-input archival tissue samples. The seamless integration with the Evosep ONE LC system by centrifugation allows ‘on-the-fly’ sample clean-up, particularly pertinent for laser microdissection workflows. We validate our method in human tonsil archival tissue, where we profile proteomes of spatially-defined B-cell, T-cell, and epithelial microregions of 4000 μm2 to a depth of ∼2000 proteins and with high cell type specificity. We finally provide detailed equipment templates and experimental guidelines for broad accessibility.

A Highly Sensitive XNA-Based RT-qPCR Assay for the Identification of ALK, RET, and ROS1 Fusions in Lung Cancer.

Lung cancer is often triggered by genetic alterations that result in the expression of oncogenic tyrosine kinases. Specifically, ALK, RET, and ROS1 chimeric receptor tyrosine kinases are observed in approximately 5-7%, 1-2%, and 1-2% of NSCLC patients, respectively. The presence of these fusion genes determines the response to tyrosine kinase inhibitors. Thus, accurate detection of these gene fusions is essential in cancer research and precision oncology. To address this need, we have developed a multiplexed RT-qPCR assay using xeno nucleic acid (XNA) molecular clamping technology to detect lung cancer fusions. This assay can quantitatively detect thirteen ALK, seven ROS1, and seven RET gene fusions in FFPE samples. The sensitivity of the assay was established at a limit of detection of 50 copies of the synthetic template. Our assay has successfully identified all fusion transcripts using 50 ng of RNA from both reference FFPE samples and cell lines. After validation, a total of 77 lung cancer patient FFPE samples were tested, demonstrating the effectiveness of the XNA-based fusion gene assay with clinical samples. Importantly, this assay is adaptable to highly degraded RNA samples with low input amounts. Future steps involve expanding the testing to include a broader range of clinical samples as well as cell-free RNAs to further validate its applicability and reliability.

Single-stranded linear amplification library preparation method for highly sensitive and low input amount ctDNA detection.

e15096 Background: Detection of circulating tumor DNA (ctDNA) or minimum residual disease (MRD) through somatic variant identifications in plasma samples has emerged as a promising approach for monitoring the clinical outcomes after surgical resections or other therapies. However, due to very low ctDNA levels in the early-stage cancers and tumor remissions after therapies, or low plasma volumes and DNA degradation caused low qualities in clinical practice, to sensitively identify somatic mutations with low allele frequencies in those samples remains a challenging technology task. Methods: In this study, we developed a single-stranded, linear amplification based library preparation sequencing (SLA-Seq) method. In the experimental workflow, double stranded molecular templates will be converted into single strands to construct libraries, thus minimize the loss of irregular double stranded DNA molecules. Adapter ligation was carried out in two steps. During the first step, P7 adapters with unique molecular identifiers(UMI) were ligated to the 3’ end of the molecular templates, and the DNA molecules were amplified by linear amplification to improve the DNA utilization efficiency. In the second step, P5 adapters were ligated to the linear amplified PCR products, then amplified by the pre-PCR. The pre-library was enriched by hybridization and magnetic-beads capturing and then subjected to high-throughput sequencing. 30ng of the cfDNA reference standard samples containing mutations of 0.3% and 0.1% allele frequencies were used to validate the performance. Results: In the SLA-Seq method, adapter sequences were carefully optimized to eliminate adapter dimers and thus improved the DNA ligation efficiency. Compared with the double-stranded library preparation method, SLA-seq showed overall higher effective depths after removing duplications if the same start amount of the DNA was used, which indicated that the higher DNA utilization rate was achieved. To evaluate the performance, we applied SLA-Seq on 11 reference standard samples with only 30ng start amount DNA. Among them, 6 samples were diluted to 0.3% allele frequency and 5 samples were diluted to 0.1% allele frequency. Totally they had 24 and 35 mutations, respectively. The sensitivity at the 0.3% allele frequency was 100% (24/24) and it was 94.29% (33/35) at the 0.1% mutation level, which also showed the higher sensitivity achieved by SLA-Seq than double-stranded library preparation method. Conclusions: We developed a single-stranded, linear amplification based library preparation method to improve difficult sample situations such as low input amount and low qualities, and achieved high single site sensitivity at the 0.1% allele frequency. Such features imply SLA-Seq has important clinical significance in the ctDNA/MRD detection.

Site-specific analysis of ribosomal 2'O-methylation by quantitative reverse transcription PCR under low deoxynucleotide triphosphate concentrations.

Ribose 2'O-methylation (Nm, ribomethylation) is the most abundant RNA modification present in rRNA. It has been shown that alterations in ribosomal 2'O-methylation at individual Nm sites likely reflect regulated cellular processes. Although several analytical approaches for Nm detection and profiling have been developed, a simple and affordable method for the screening and measurement of individual Nm sites in large numbers of tissue samples is required to examine their potential for clinical translation. Here, we describe a new quantitative reverse transcription PCR-based method that can sensitively assess ribomethylation levels at specific rRNA sites at single-nucleotide resolution in low input amounts of total RNA.

NanoNIPT: Short-fragment nanopore sequencing of cell-free DNA for non-invasive prenatal testing of fetal aneuploidies and sex chromosome aberrations.

N/A This article is protected by copyright. All rights reserved.

Quality Control of Biospecimens in a Danish Clinical Cytology Biobank.

Introduction: Based on the experience from a Swedish biobank, we established a clinical cervical cytology biobank and adapted it to a Danish setting. The aim of the present study was to validate the biobank material regarding quality and quantity, to determine the usefulness of the material for future diagnostics and biomarker testing. Methods: Cervical cytology samples collected in ThinPrep were analyzed before and after biobanking using p16/ki-67 dual staining, a human papillomavirus (HPV) DNA test (Cobas), and a test for HPV messenger RNA (mRNA; Aptima). The concordance of the test results before and after biobanking was assessed. We also evaluated the morphology before and after biobanking and did additional tests on the biobanked material to qualify the usefulness of the material (library preparation for next-generation sequencing [NGS], reverse transcription-polymerase chain reaction [RT-PCR], and the Inno-Lipa HPV genotyping test). Results: For the Cobas HPV test, the concordance was 92% (122/133), and for the Inno-Lipa test (30 samples), it was 100%. For the Aptima assay, the concordance was a little lower, 84% (42/50). The morphology of the cell was well preserved, and the concordance of the p16/ki-67 dual staining was 88% (37/42). The functional tests showed that DNA-based NGS libraries (TST15 panel; Illumina) had good quality parameters. However, with the RT-PCR, 12% of the samples showed poor quality and a too low input amount for the analysis. Conclusion: The quality of the biobanked samples is high, and the material is suitable for testing of DNA, RNA, and protein. However, for testing of specific biomarkers, pilot studies are recommended to ensure sufficient input amount and quality of the material, especially for RNA-based studies.

Abstract 5784: Overcoming FFPE hurdles to enable high quality hybrid capture libraries and somatic mutation detection in matched tumor-normal patient samples

Abstract In cancer genomics, a common source of DNA is formalin-fixed, paraffin-embedded (FFPE) tissue from patient surgical samples, where in most cases high quality fresh or frozen tissue samples are not available. FFPE DNA poses many notable challenges for preparing NGS libraries, including low input amounts and highly variable damage from fixation, storage, and extraction methods. Due to the high cost of sequencing and variability of coverage, regions of interest are often specifically enriched using hybrid capture-based approaches, but these methods require a high input of diverse, uniform DNA library to achieve the coverage required for somatic mutation identification in tumor samples.We applied a multi-faceted approach to improving the yield, quality, and coverage from libraries generated from cancer patient sample sets. Using tumor-normal pairs, we evaluated the impact of our method on the sensitivity and accuracy of somatic variant detection. Matched frozen tissue provided a truth set of somatic variants for a particular tumor as well as a gold standard by which we could assess the quality of our FFPE hybrid capture libraries. Combining DNA damage repair and a novel enzymatic fragmentation mix upstream of library preparation not only reduced the false positive rate in somatic variant detection by repairing damage-derived mutations but also improved the library yield, complexity, coverage uniformity, and hybrid capture library quality metrics. The use of unique molecular identifier (UMI)-containing adaptors ensured accurate duplicate marking. Finally, a new PCR master mix boosts the library yield without compromising library quality in FFPE samples ranging from very poor quality to high quality. This combinatorial approach allows even the poorest quality FFPE samples to achieve high quality libraries with sufficient input for hybrid capture in our study. Citation Format: Margaret R. Heider, Jian Sun, Brittany S. Sexton, Bradley W. Langhorst, Andrew Gray, Lauren Higgins, Lixin Chen, Lynne Apone, Thomas C. Evans, Nicole M. Nichols, Eileen T. Dimalanta, Theodore B. Davis, Pingfang Liu. Overcoming FFPE hurdles to enable high quality hybrid capture libraries and somatic mutation detection in matched tumor-normal patient 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 5784.

DdPCR allows 16S rRNA gene amplicon sequencing of very small DNA amounts from low-biomass samples

BackgroundOne limiting factor of short amplicon 16S rRNA gene sequencing approaches is the use of low DNA amounts in the amplicon generation step. Especially for low-biomass samples, insufficient or even commonly undetectable DNA amounts can limit or prohibit further analysis in standard protocols.ResultsUsing a newly established protocol, very low DNA input amounts were found sufficient for reliable detection of bacteria using 16S rRNA gene sequencing compared to standard protocols. The improved protocol includes an optimized amplification strategy by using a digital droplet PCR. We demonstrate how PCR products are generated even when using very low concentrated DNA, unable to be detected by using a Qubit. Importantly, the use of different 16S rRNA gene primers had a greater effect on the resulting taxonomical profiles compared to using high or very low initial DNA amounts.ConclusionOur improved protocol takes advantage of ddPCR and allows faithful amplification of very low amounts of template. With this, samples of low bacterial biomass become comparable to those with high amounts of bacteria, since the first and most biasing steps are the same. Besides, it is imperative to state DNA concentrations and volumes used and to include negative controls indicating possible shifts in taxonomical profiles. Despite this, results produced by using different primer pairs cannot be easily compared.

Abstract 455: Consistent multi-lab performance of highly multiplexed RNA fusion detection with low RNA input and limit of detection

Abstract Gene fusions, which are usually caused by chromosomal rearrangements, are frequently associated with various cancers. Detection of fusion mutations is an important part of cancer management, and are usually detected in RNA purified from biopsies of fresh tissue or FFPE sections. The amount of RNA from these samples, however, is usually limited. We developed a new, PCR-based fusion detection method, which even allows detection of unknown and novel fusion mutations. We demonstrate its consistent performance across various RNA inputs, and sequencing depths, in terms of technical performance and limit of detection. A multiplex PCR RNA fusion panel was designed to cover known RNA fusion targets and unknown fusion targets for target enrichment via single primer multiplex PCR. SeraSeq fusion Reference RNA was used to evaluate the limit of detection of the method. The amplicons were about 110 bp on average to allow for target amplification from challenging fragmented samples such as FFPE RNA. After reverse transcription, the panel was used in a multiplex PCR to amplify the targets, and a final PCR was used to add sequencing adapters and sample indexes. The resulting libraries were sequenced in Illumina sequencers. The library was analyzed for the detection of known fusion variants across different initial input of targets, as well as the mapping rate, onto the target, and the presence of human ribosomal RNA. We found that the lengths of the fragmented RNA had little effect on the performance of the libraries. With various amount of input RNA, the required numbers of PCR cycles were optimized. We observe a linear relationship between the library yields and input amounts of RNA from 10 to 50ng, which had R1 mapping rates above 95% and 98%, respectively. We report the detection of all twelve fusion variants, which are present in the panel and in the Seraseq Fusion RNA Mix v4 reference standard and covered by our panel on each NGS platform with an average sequencing depth of 0.2M reads, or 3600 reads/amplicon across all assays. Even at inputs as low as 3 ng, all 12 fusions were typically detected. In general, the results for individual fusions among the different replicates were concordant, with limited observed variance in reads across some fusion junctions between assays and replicates. Over 90% of reads supported the fusion call from all inputs (ranging from 1.6 to 50 ng). Over 97% of reads supported the fusion call between 12 and 50 ng. A multi-lab validation confirmed the above results, and were also comparable when using an additional distinct CleanPlex fusion panel. CleanPlex fusion technology allows for reliable and simultaneous detection of 12 designed clinically relevant RNA fusions even at low input amounts. The data from a multi-lab study support the use of this technology for targeted NGS RNA fusion assays. The CleanPlex fusion technology provides consistent and sensitive results for a wide variety of sample inputs. Citation Format: David N. Debruyne, Chenyu Li, Julia Spencer, Vidushi Kapoor, Lily Y. Liu, Lucie Lee, Rounak Feigelman, Grayson Burdon, Jeffrey Liu, Guoying Liu, Zhitong Liu. Consistent multi-lab performance of highly multiplexed RNA fusion detection with low RNA input and limit of detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 455.

Abstract 542: Validation of low fraction allelic variants in plasma samples from patients with late stage colorectal cancer using droplet digital PCR: Potential clinical utility for minimal residual disease monitoring

Abstract Introduction: Reliable detection of low mutant allele fractions (MAF) in circulating tumor DNA (ctDNA) offers clinically crucial information. Importantly, minimal residual disease (MRD) detection in solid tumors has been recognized as a powerful readout for response and early relapse prediction. Considering the high sample input requirement and assay complexity of whole exome sequencing (WES) or targeted NGS technologies, it is essential to develop a cost-effective absolute quantification approach to measure MAF with low DNA input amount. Clinically relevant gene variants present both in plasma ctDNA and tissue of origin were identified in a previous study of 25 late-stage CRC samples analyzed using the Guardant Health GuardantOMNI NGS panel (ctDNA) and WES (tumor). We further investigated the potential of using droplet digital PCR (ddPCR) to measure variants present only in ctDNA; particularly for those at low MAF which could be applied for MRD monitoring. Methods: We developed ddPCR assays for 13 variants which were detected with the GuardantOMNI panel in ctDNA and not tissue from 5 of 25 advanced stage CRC samples. MAF for the previously reported variants ranged from 64.0% (FBXW7 p.R505C) to 1.2% (TP53 R273H). Among the newly investigated variants the range was 2.2% (EPHA7 I146T) to 0.4% (PDGFRA1 pL31F and RB1 p.E440K). Other variants included KRAS G12D (30.8%), PIK3CA p.C420R (29.2%), TP53 p.R273H (1.2%) and ERBB4 p.I736T (1.1%). Positive control cDNA was designed and spiked into a pool of healthy plasma to determine the baseline wildtype levels and the sensitivity of each assay. Patient ctDNA was then measured. Results: MAF from ddPCR data and the GuardantOMNI panel showed high concordance (r2 = 0.9986) for all data points (n=13). When only considering those variants with MAF less than 5% (n=9; TP53 R273H, TSC2, EP300, PPP2R1A, LIG4, EPHA7, ERBB4, PDGFRA1, and RB1) the concordance remained high with r2 = 0.9532, thus demonstrating the accuracy and sensitivity of both platforms. All ddPCR results were achieved with at most 5ng input DNA and as low as 1ng in most cases. However, the concordance begins to weaken below 1% MAF (r2=0.4024), likely due to the limit of detection of the current ddPCR assays. Conclusions: We demonstrated that ddPCR offers a highly sensitive and purely quantitative method to measure low MAF with minimal sample input requirements, which highlights the potential use of ddPCR for MRD monitoring. As one possible MRD monitoring approach, NGS panel-based assays may identify all variants at baseline screening, followed by ddPCR as a complementary solution for MRD monitoring of single variants. Currently, there are ongoing efforts in examining longitudinal ctDNA variant changes in CRC patients receiving treatment to further confirm which variants could be used for MRD monitoring in CRC. Citation Format: Dennis O'Rourke, Danyi Wang, Juergen Scheuenpflug, Zheng Feng. Validation of low fraction allelic variants in plasma samples from patients with late stage colorectal cancer using droplet digital PCR: Potential clinical utility for minimal residual disease monitoring [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 542.

Evaluation of whole-genome DNA methylation sequencing library preparation protocols

BackgroundWith rapidly dropping sequencing cost, the popularity of whole-genome DNA methylation sequencing has been on the rise. Multiple library preparation protocols currently exist. We have performed 22 whole-genome DNA methylation sequencing experiments on snap frozen human samples, and extensively benchmarked common library preparation protocols for whole-genome DNA methylation sequencing, including three traditional bisulfite-based protocols and a new enzyme-based protocol. In addition, different input DNA quantities were compared for two kits compatible with a reduced starting quantity. In addition, we also present bioinformatic analysis pipelines for sequencing data from each of these library types.ResultsAn assortment of metrics were collected for each kit, including raw read statistics, library quality and uniformity metrics, cytosine retention, and CpG beta value consistency between technical replicates. Overall, the NEBNext Enzymatic Methyl-seq and Swift Accel-NGS Methyl-Seq kits performed quantitatively better than the other two protocols. In addition, the NEB and Swift kits performed well at low-input amounts, validating their utility in applications where DNA is the limiting factor.ResultsThe NEBNext Enzymatic Methyl-seq kit appeared to be the best option for whole-genome DNA methylation sequencing of high-quality DNA, closely followed by the Swift kit, which potentially works better for degraded samples. Further, a general bioinformatic pipeline is applicable across the four protocols, with the exception of extra trimming needed for the Swift Biosciences’s Accel-NGS Methyl-Seq protocol to remove the Adaptase sequence.

Consistent performance of highly multiplexed RNA fusion detection with low RNA input and limit of detection.

e15029 Background: Gene fusions are usually caused by chromosomal rearrangements and frequently associated with various cancers. Detection of fusion mutations is an important part of cancer management, and are usually detected in RNA purified from biopsies of fresh tissue or FFPE sections. However, the amount of RNA from these samples is usually limited. We developed a new, PCR-based fusion detection method, which allows detection of unknown and novel fusion mutations. We demonstrate its consistent performance across various RNA inputs, and sequencing depths, in terms of technical performance and limit of detection. Methods: A multiplex PCR RNA fusion panel was designed to cover known RNA fusion targets and unknown fusion targets for target enrichment via single primer multiplex PCR. SeraSeq fusion Reference RNA was used to evaluate the limit of detection of the method. The amplicons were about 110 bp on average to allow for target amplification from challenging fragmented samples such as FFPE RNA. After reverse transcription, the panel was used in a multiplex PCR to amplify the targets, and a final PCR was used to add sequencing adapters and sample indexes. The resulting libraries were sequenced on Illumina sequencers. The data was analyzed for the detection of known fusion variants across varying initial inputs of targets, as well as the performance in terms of mapping and on-target rates, as well as the presence of human ribosomal RNA. Results: We found that the fragmented RNA length had little effect on the performance of the libraries. The required numbers of PCR cycles were optimized based on testing with various amounts of starting RNA input material. We observe a linear relationship between the library yields and RNA input amounts from 10 to 50ng, which had R1 mapping rates above 95% and 98%, respectively. We report the detection of all twelve fusion variants, which are targeted by our panel and present in the Seraseq Fusion RNA Mix v4 reference standard, and which are covered with an average sequencing depth of 0.2M reads, or 3600 reads/amplicon, across all assays. Even at inputs as low as 3 ng, all 12 fusions were typically detected. In general, the results for individual fusions among the different replicates were concordant, with limited observed variance in reads across some fusion junctions between assays and replicates. Over 90% of the reads supported the fusion calls from all inputs (ranging from 1.6 to 50 ng). Over 97% of the reads supported the fusion calls between 12 and 50 ng. Conclusions: A multi-lab validation confirmed the above results, and were also comparable when using an additional distinct CleanPlex fusion panel. To conclude, our CleanPlex fusion technology allows for reliable and simultaneous detection of 12 designed clinically relevant RNA fusions even at low input amounts.

Abstract 3619: High quality library preparation and accurate sequencing from highly damaged matched patient sample sets

Abstract Preparation of high quality next-generation sequencing (NGS) libraries is critical for accurate identification of disease-causing mutations, but clinical scientists are often limited by the input DNA quality. In cancer genomics, a common source of DNA is formalin-fixed, paraffin-embedded (FFPE) tissue from patient biopsy, and in most cases high quality fresh or frozen tissue samples are not available. FFPE DNA poses many notable challenges for preparing NGS libraries, including low input amounts and extensive damage from fixation, storage, and extraction methods. Sequencing artifacts arising from damaged DNA bases raise the level of apparent mutations, making it more difficult to discern true, low frequency, disease-related variants from noise. We developed a second-generation, multi-enzyme DNA repair mix (V2) that is designed to repair the most prevalent damage types found in FFPE DNA including cytosine deamination, oxidative damage, abasic sites, nicks, and gaps. We tested the application of the V2 enzymatic repair mix in Illumina library preparation for DNA samples extracted from a series of matched patient sample sets, which included: FFPE normal and tumor tissue, and frozen normal and tumor tissue for each patient. Following this, we performed target enrichment, deep sequencing, and variant calling analyses. Our library preparation workflow integrates enzymatic repair upstream of Illumina library preparation without an additional bead purification step, maximizing library yield and complexity from precious FFPE samples. Rescue of damaged DNA molecules for library conversion and repair of damaged DNA bases improved on-target rate, coverage uniformity, and duplication rates. With a matched frozen sample for each FFPE specimen, we demonstrate that repair of damaged DNA bases improves sequencing accuracy by reducing the number of false-positive variant calls. We propose that enzymatic repair enables researchers to capture as much useable, accurate data from poor quality FFPE samples as possible. Citation Format: Margaret R. Heider. High quality library preparation and accurate sequencing from highly damaged matched patient sample sets [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 3619.

Rapid Next-Generation Sequencing–Based Diagnostics of Bacteremia in Septic Patients

The increasing incidence of bloodstream infections including sepsis is a major challenge in intensive care units worldwide. However, current diagnostics for pathogen identification mainly depend on culture- and molecular-based approaches, which are not satisfactory regarding specificity, sensitivity, and time to diagnosis. Herein, we established a complete diagnostic workflow for real-time high-throughput sequencing of cell-free DNA from plasma based on nanopore sequencing for the detection of the causative agents, which was applied to the analyses of eight samples from four septic patients and three healthy controls, and subsequently validated against standard next-generation sequencing results. By optimization of library preparation protocols for short fragments with low input amounts, a 3.5-fold increase in sequencing throughput could be achieved. With tailored bioinformatics workflows, all eight septic patient samples were found to be positive for relevant pathogens. When considering time to diagnosis, pathogens were identified within minutes after start of sequencing. Moreover, an extrapolation of real-time sequencing performance on a cohort of 239 septic patient samples revealed that more than 90% of pathogen hits would have also been detected using the optimized MinION workflow. Reliable identification of pathogens based on circulating cell-free DNA sequencing using optimized workflows and real-time nanopore-based sequencing can be accomplished within 5 to 6 hours following blood draw. Therefore, this approach might provide therapy-relevant results in a clinically critical timeframe.

Abstract 1709: Consistent performance of highly multiplexed RNA fusion reference materials across different NGS assays in a multi-lab study

Abstract Introduction: Gene fusions, usually the result of chromosomal rearrangements, are frequently associated with many cancer types, and hence clinically actionable, making fusion detection an important part of cancer disease management. We developed a new, optimized version of the Seraseq® Fusion RNA Reference Material, and demonstrate its consistent performance across different NGS enrichment assays, sequencing coverage depths, bioinformatics pipelines, and RNA mass inputs from a multi-lab investigation (Sites A, B, C, D & E). Methods: Biosynthetic DNA was used for transcription of 18 RNA fusions, including more common fusions of ALK, RET, and ROS1, as well as rare fusion events such as PAX-PPARG and ETV6-NTRK3. The in vitro transcribed RNAs were mixed with total RNA extracted from GM24385 reference cell line (The 1000 Genomes Project, Coriell). Digital PCR with TaqMan® chemistry was used to determine the target fusion RNA concentration and serve as the “truth” data set for comparison to NGS, which can be variable depending on input, assay, and bioinformatics. The fusion-total RNA mix was analyzed by five external laboratories; it was tested using the ArcherDx FusionPlex™ Solid Tumor Panel (Site A, Site B, Site C), the ArcherDx FusionPlex™ CTL Panel (Site C), the ArcherDx FusionPlex™ Lung Panel (Site B), a custom ArcherDx FusionPlex™ Panel (Site D), and the TruSight Tumor 170 Panel (Site E). Results: All eighteen (18) fusions in the new Seraseq Fusion RNA Mix v4 reference standard were detected as expected on each NGS platform with an average of greater than 85% of on-target reads across all assays. Even at inputs as low as 20 ng, all 18 fusions were typically detected above fusion-calling thresholds. In general, the results for individual fusions among the different NGS panels and among replicates were concordant, with observed variance in reads across some fusion junctions between assays and replicates. Within FusionPlex assay results, the average percent of reads supporting the fusion call across all fusions was about 63%, regardless of input (a range between 20 to 250 ng). Collectively, the multi-lab results confirm that the Seraseq® Fusion RNA Mix v4 reference standard is compatible with both amplicon and hybridization-capture based NGS assays. Conclusions: Seraseq RNA Fusion Mix v4 has broad NGS assay compatibility and allows for reliable and simultaneous detection of 18 clinically relevant RNA fusions even at low input amounts. The data from a multi-lab study support the use of this reference standard for targeted NGS assay development, assay validation, bioinformatics pipeline optimization, and as positive controls in clinical NGS RNA fusion assays. The biosynthetic manufacturing approach produces reference materials that provide consistent results for a wide variety of common and rare gene fusions. Citation Format: Dana J. Ruminski Lowe, Deepika Philkana, Catherine Huang, Omoshile Clement, Andrew Anfora, Dan Brudzewsky, Bharathi Anekella. Consistent performance of highly multiplexed RNA fusion reference materials across different NGS assays in a multi-lab study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1709.

Systematic analysis of TruSeq, SMARTer and SMARTer Ultra-Low RNA-seq kits for standard, low and ultra-low quantity samples

High-throughput RNA-sequencing has become the gold standard method for whole-transcriptome gene expression analysis, and is widely used in numerous applications to study cell and tissue transcriptomes. It is also being increasingly used in a number of clinical applications, including expression profiling for diagnostics and alternative transcript detection. However, despite its many advantages, RNA sequencing can be challenging in some situations, for instance in cases of low input amounts or degraded RNA samples. Several protocols have been proposed to overcome these challenges, and many are available as commercial kits. In this study, we systematically test three recent commercial technologies for RNA-seq library preparation (TruSeq, SMARTer and SMARTer Ultra-Low) on human biological reference materials, using standard (1 mg), low (100 ng and 10 ng) and ultra-low (<1 ng) input amounts, and for mRNA and total RNA, stranded and unstranded. The results are analyzed using read quality and alignment metrics, gene detection and differential gene expression metrics. Overall, we show that the TruSeq kit performs well with an input amount of 100 ng, while the SMARTer kit shows decreased performance for inputs of 100 and 10 ng, and the SMARTer Ultra-Low kit performs relatively well for input amounts <1 ng. All the results are discussed in detail, and we provide guidelines for biologists for the selection of an RNA-seq library preparation kit.

OncoKids: A Comprehensive Next-Generation Sequencing Panel for Pediatric Malignancies

The OncoKids panel is an amplification-based next-generation sequencing assay designed to detect diagnostic, prognostic, and therapeutic markers across the spectrum of pediatric malignancies, including leukemias, sarcomas, brain tumors, and embryonal tumors. This panel uses low input amounts of DNA (20 ng) and RNA (20 ng) and is compatible with formalin-fixed, paraffin-embedded and frozen tissue, bone marrow, and peripheral blood. The DNA content of this panel covers the full coding regions of 44 cancer predisposition loci, tumor suppressor genes, and oncogenes; hotspots for mutations in 82 genes; and amplification events in 24 genes. The RNA content includes 1421 targeted gene fusions. We describe the validation of this panel by using a large cohort of 192 unique clinical samples that included a wide range of tumor types and alterations. Robust performance was observed for analytical sensitivity, reproducibility, and limit of detection studies. The results from this study support the use of OncoKidsfor routine clinical testing of a wide variety of pediatric malignancies.

Abstract 1425: A multi-enzyme DNA repair mix improves library quality and sequencing accuracy in FFPE tumor samples

Abstract Next-generation sequencing (NGS) methods are used extensively to profile mutations present in diseased human tissues. These genomic approaches hold great promise for personalized medicine but sequencing accuracy is essential for proper patient diagnosis and determining a treatment plan. A common source of DNA for genomic profiling is formalin-fixed, paraffin-embedded (FFPE) tissue samples obtained from patient biopsy. FFPE DNA poses important challenges for preparing NGS libraries including low input amounts and poor DNA quality, resulting from extensive fixation- and storage-induced DNA damage. Additionally, these damage-induced sequencing artifacts raise the background level of mutations, making it difficult to discern true, low frequency, disease-causing variants from noise. We previously showed that a major fraction of somatic mutations described in publicly available datasets are due to such sequencing artifacts (Chen et al., Science 2017). Furthermore, we showed that enzymatic repair of DNA before library preparation improves the library quality and reduces background noise. We developed a second-generation DNA repair enzyme mix (V2) that efficiently repairs the most prevalent damage types found in FFPE DNA and further improves the quality and yield of NGS libraries. Additionally, we tested the efficacy of the V2 repair mix in improving sequencing accuracy for FFPE DNA samples obtained from different cancer tissues. We performed target enrichment on a panel of 151 cancer genes, deep sequenced, and performed variant analysis. For a subset of variants, we further validated our results using a droplet digital PCR (ddPCR) assay. Both methods showed that the V2 repair mix did not alter the overall frequency of variants identified, thus it did not introduce bias, but significantly improved the sequencing accuracy by reducing the number of false variant calls. Therefore, enzymatic repair is a critical first step in preparing FFPE DNA sequencing libraries, allowing more sensitive and robust detection of low frequency, disease variants. Citation Format: Pingfang Liu, Margaret Heider, Chen Song, Lixin Chen, Laurence Ettwiller, Lauren Higgins, Eileen Dimalanta, Theodore Davis, Thomas Evans. A multi-enzyme DNA repair mix improves library quality and sequencing accuracy in FFPE tumor samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1425.