Amount Of Input DNA Research Articles

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.

Target enrichment of metazoan mitochondrial DNA with hybridization capture probes

High-throughput DNA sequencing studies allow rapid and widescale investigations into complex assemblages of organisms that would otherwise remain infeasible. Recently scientists have begun to apply metagenomic sequencing (sequencing of the complete pool of DNA from a sample) to investigate microscopic animal communities. Mitochondrial genomes are an important locus for such studies; however, current databases of complete mitochondrial genomes remain incomplete, and many important meiofaunal clades are underrepresented or missing entirely. This underrepresentation is likely due to the low amount of input DNA acquired from meiofaunal individuals, the impossibility of pooling animals because of high levels of cryptic speciation, and the paucity of researchers collecting and studying these animals. Here we help to alleviate the first two barriers by developing a method of enriching animal mtDNA from next-generation sequencing libraries by using hybridization capture probes. These hybrid-capture probes were designed based on the complete collection of mitochondrial genomes available from GenBank and are suitable for enrichment of diverse animal lineages. We demonstrate the protocol on forty-seven meiofaunal specimens spanning four different phyla. Overall, we observed an average of ~450× fold enrichment over standard NGS sequencing library preparation. In addition, we were able to enrich the mtDNA from taxa distantly related to those in the database, indicating that even animals that were not directly targeted by the probe design can be enriched with this method.

Quantification of DNA methylation independent of sodium bisulfite conversion using methylation-sensitive restriction enzymes and digital PCR.

Epigenetic regulation is important in human health and disease, but the exact mechanisms remain largely enigmatic. DNA methylation represents one epigenetic aspect but is challenging to quantify. In this study, we introduce a digital approach for the quantification of the amount and density of DNA methylation. We designed an experimental setup combining efficient methylation‐sensitive restriction enzymes with digital polymerase chain reaction (PCR) to quantify a targeted density of DNA methylation independent of bisulfite conversion. By using a stable reference and comparing experiments treated and untreated with these enzymes, copy number instability could be properly normalized. In silico simulations demonstrated the mathematical validity of the setup and showed that the measurement precision depends on the amount of input DNA and the fraction methylated alleles. This uncertainty could be successfully estimated by the confidence intervals. Quantification of RASSF1 promoter methylation in a variety of healthy and malignant samples and in a calibration curve confirmed the high accuracy of our approach, even in minute amounts of DNA. Overall, our results indicate the possibility of quantifying DNA methylation with digital PCR, independent of bisulfite conversion. Moreover, as the context‐density of methylation can also be determined, biological mechanisms can now be quantitatively assessed.

Improved STR analysis of degraded DNA from human skeletal remains through in-house MPS-STR panel.

DNA analysis of degraded samples and low-copy number DNA derived from skeletal remains, one of the most challenging forensic tasks, is common in disaster victim identification and genetic analysis of historical materials. Massively parallel sequencing (MPS) is a useful technique for STR analysis that enables the sequencing of smaller amplicons compared with conventional capillary electrophoresis (CE), which is valuable for the analysis of degraded DNA. In this study, 92 samples of human skeletal remains (70+ years postmortem) were tested using an in-house MPS-STR system designed for the analysis of degraded DNA. Multiple intrinsic factors of DNA from skeletal remains that affect STR typing were assessed. The recovery of STR alleles was influenced more by DNA input amount for amplification rather than DNA degradation, which may be attributed from the high quantity and quality of libraries prepared for MPS run. In addition, the higher success rate of STR typing was achieved using the MPS-STR system compared with a commercial CE-STR system by providing smaller sized fragments for amplification. The results can provide constructive information for the analysis of degraded sample, and this MPS-STR system will contribute in forensic application with regard to skeletal remain sample investigation.

Abstract 2489: Characterization of breast cancer ctDNA with Omi-Seq

Abstract Cancer patients' cell free DNA (cfDNA) contains circulating tumor-derived DNA (ctDNA). The fast dynamic of clearance allows ctDNA to be used as biomarkers for tumor real time profiling. The benefit of non-invasive, cost effective, and easily-repetitive sampling makes liquid biopsy a highly-desirable option for molecular assessment. Deep sequencing has been widely used to investigate subpopulations in complex biological samples. However, there are still limitations of the sequencing technology due to the low yield of cfDNA amount, and errors introduced during sample preparation and sequencing. Approximately 1% of bases are incorrectly identified, depending on the specific platform and sequence context. Although Molecular Identifier can theoretically greatly reduce errors, it suffers from several drawbacks. First, it is difficult to synthesize double strand adapters with randomized Molecular Identifier. Second, due to the difficulty in making high quality Single Molecular Identifier (SMI) adaptors, the ligation efficiency might be compromised and therefore might require large amounts of input DNA. To optimize the liquid biopsy protocol, we first examined if there is any bias in the molecular barcodes' selection during library preparation. Indeed, a skewed trend with molecular barcode selection in ligation process has been detected. We also evaluated the error rates in molecular barcodes and found that there is a substantial amount of errors (~5% of total reads carrying the wrong molecular barcode). To overcome these issues, Omi-seq calculates Digital Molecular Identifier (DMI) using a pool of regular adapters with defined barcodes which can be easily synthesized in high quality. We then analyzed Horizon multiplex I cfDNA reference standard with the lowest available allele frequency (AF) 0.1%. With Omi-error correction, Omi-Seq was able to reproducibly detect the variants. We also evaluated Omi-Seq in real patient samples, Omi-Seq is able to detect variants at AF 0.03%. Omi-Seq was utilized to created cancer profiles for 202 subjects enrolled in The First Affiliated Hospital of Wenzhou Medical University breast cancer study. The gene landscape indicated that the most frequently detected genes are PIK3CA and TP53, followed by ERBB2. The accuracy of Her2 CNV in tissue/FFPE sample is 96% with 85 % sensitivity and 100% specificity. Mutation numbers vary between different molecular subtypes of cfDNA, where the basal-like group had more variants detected than Luminal patients. Furthermore, the ratio changes of max ctDNA AF (ctF) are highly correlated to the clinical response measurement, including cancer relapse, metastatic and treatment resistance. Our data show that ctDNA characterization with Omi-seq could extend the capacity of personalized cancer clinical management. Citation Format: Yinghao Wang, Yaoyao Guan, Jingjing Xiang, Lingguo Kong, Lizhi Lin, Judy Webb, Yunguang Tong, Ouchen Wang. Characterization of breast cancer ctDNA with Omi-Seq [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 2489.

A sequence-based 74plex microhaplotype assay for analysis of forensic DNA mixtures

Microhaplotypes are emerging biomarkers for forensic applications. In this study, a sequence-based multiplex assay of 74 microhaplotypes (230 SNPs) was developed on the Ion Torrent S5™ (Thermo Fisher Scientific) system and the potential for its application to mixture deconvolution was explored. The 74 loci are distributed across the autosomal human genome and have Ae (i.e., effective number of alleles) values ranging from 1.307 to 6.010 (median = 2.706) and In (i.e., informativeness) values ranging from 0.096 to 0.660 (median = 0.251); the amplicon sizes range between 157 and 325 bp. The typing performance of the panel was evaluated on a series of in-silico two to five-person DNA mixtures and results were compared to fragment and sequence-based STRs. The 74plex-locus assay was found sensitive down to 0.05 ng of input DNA and effective for the analysis of mixtures at different contributor ratios and input DNA amounts. As expected, none or very partial minor CE-STR profile(s) were reported for highly imbalanced two-person and high-order DNA mixtures while sequencing of STRs enabled the detection of more individual minor alleles. For microhaplotypes, a full minor profile was detected down to a 20:1 ratio at 10 ng and minimal allele dropout at 1 ng of input DNA. A higher rate of allele dropout from the minor donor(s) was reported at 1 ng than 10 ng for three-person mixtures while for four- and five-person mixtures, the same number of dropouts was observed for almost all minor donors. Overall this microhaplotype panel is a powerful tool that can complement and enhance size- and sequence-based STR analysis of forensic DNA mixtures.

Design and Testing of a Custom Melanoma Next Generation Sequencing Panel for Analysis of Circulating Tumor DNA.

Detection of melanoma-associated mutations using circulating tumor DNA (ctDNA) from plasma is a potential alternative to using genomic DNA from invasive tissue biopsies. In this study, we developed a custom melanoma next-generation sequencing (NGS) panel which includes 123 amplicons in 30 genes covering driver and targetable mutations and alterations associated with treatment resistance. Analysis of a cohort of 74 stage III and IV treatment-naïve melanoma patients revealed that sensitivity of ctDNA detection was influenced by the amount of circulating-free DNA (cfDNA) input and stage of melanoma. At the recommended cfDNA input quantity of 20 ng (available in 28/74 patients), at least one cancer-associated mutation was detected in the ctDNA of 84% of stage IV patients and 47% of stage III patients with a limit of detection for mutant allele frequency (MAF) of 0.2%. This custom melanoma panel showed significant correlation with droplet digital PCR (ddPCR) and provided a more comprehensive melanoma mutation profile. Our custom panel could be further optimized by replacing amplicons spanning the TERT promoter, which did not perform well due to the high GC content. To increase the detection rate to 90% of stage IV melanoma and decrease the sensitivity to 0.1% MAF, we recommend increasing the volume of plasma to 8 mL to achieve minimal recommended cfDNA input and the refinement of poorly performing amplicons. Our panel can also be expanded to include new targetable and treatment resistance mutations to improve the tracking of treatment response and resistance in melanoma patients treated with systemic drug therapies.

Evaluation of Experimental Protocols for Shotgun Whole-Genome Metagenomic Discovery of Antibiotic Resistance Genes.

Shotgun metagenomics has enabled the discovery of antibiotic resistance genes (ARGs). Although there have been numerous studies benchmarking the bioinformatics methods for shotgun metagenomic data analysis, there has not yet been a study that systematically evaluates the performance of different experimental protocols on metagenomic species profiling and ARG detection. In this study, we generated 35 whole genome shotgun metagenomic sequencing data sets for five samples (three human stool and two microbial standard) using seven experimental protocols (KAPA or Flex kits at 50ng, 10ng, or 5ng input amounts; XT kit at 1ng input amount). Using this comprehensive resource, we evaluated the seven protocols in terms of robust detection of ARGs and microbial abundance estimation at various sequencing depths. We found that the data generated by the seven protocols are largely similar. The inter-protocol variability is significantly smaller than the variability between samples or sequencing depths. We found that a sequencing depth of more than 30M is suitable for human stool samples. A higher input amount (50ng) is generally favorable for the KAPA and Flex kits. This systematic benchmarking study sheds light on the impact of sequencing depth, experimental protocol, and DNA input amount on ARG detection in human stool samples.

One fly-one genome: chromosome-scale genome assembly of a single outbred Drosophila melanogaster.

A high quality genome assembly is a vital first step for the study of an organism. Recent advances in technology have made the creation of high quality chromosome scale assemblies feasible and low cost. However, the amount of input DNA needed for an assembly project can be a limiting factor for small organisms or precious samples. Here we demonstrate the feasibility of creating a chromosome scale assembly using a hybrid method for a low input sample, a single outbred Drosophila melanogaster. Our approach combines an Illumina shotgun library, Oxford nanopore long reads, and chromosome conformation capture for long range scaffolding. This single fly genome assembly has a N50 of 26 Mb, a length that encompasses entire chromosome arms, contains 95% of expected single copy orthologs, and a nearly complete assembly of this individual's Wolbachia endosymbiont. The methods described here enable the accurate and complete assembly of genomes from small, field collected organisms as well as precious clinical samples.

Abstract P5-01-11: Tracking anchor mutations in serial cell-free DNA using ultra-high sensitive mass spectrometry method provide risk of subsequent recurrence during surveillance after standard therapy

Abstract Cell-free DNA (cfDNA), as a non-invasive strategy, provides substantial benefit to overcome temporal/spatial tumor heterogeneity. Surveillance of recurrence after standard treatment in early breast cancer (BC) using cfDNA, enables to detect minimal residual disease (MRD), also to identify genomic alterations driving recurrences. We aimed to assess whether cfDNA can detect metastasis during surveillance after standard treatment by tracking mutations using mass spectrometry method from minimal plasma volume. Thirty breast cancer patients with serial plasma adequately drawn for cfDNA analysis were analyzed. Twenty cases were who subsequently developed clinical recurrence while ten were clinically disease free till last follow up. Primary tumor DNA extracted from paraffin blocks of surgical specimen and germline DNA were sent for deep targeted sequencing using in-house Oncopanel (382 genes, 184 hotspots, 8 fusions, 0.82Mb) Mean sequencing depth of tumor was x284.5 and somatic mutations with >5-10% variant allelic fraction (VAF) were chosen as anchor mutations for serial analyses. Hotspot mutations eg. PIK3CA H1047 or E545K were selected for serial tracking. We applied in-house developed mass stectrometry UHS-platform (Ultra-high sensitive) -detects 0.01% frequency mutant allele in colorectal and lung cancer- from plasma processed from 2ml whole blood. Mean number of anchor mutations is 5.95 and 74.6% of mutation sites were successfully designed as multiplex-probe for UHS-platform. Anchor mutations were searched in each patients’ blood drawn every 3-6months after surgery or after time of recurrence. Among 20 patients who were clinically diagnosed to have recurrence after standard treatment, 14 displayed ctDNA positive of at least one of anchor mutations (sensitivity, 70%, false negative rate (FNR) 30%). Sensitivity was not associated with input DNA amount nor with disease free interval. It was not associated with whether patients were receiving systemic therapy at time of recurrence. Among the 6 false negative cases, 3 cases were local/or regional recurrences and the other three had distant metastasis (2 lung, 1 bone metastasis). The FNR was 0% (sensitivity 100%) however, when number of anchor mutations was more than 5. Among 10 patients with no evidence of recurrence after standard therapy, 1 patient had cfDNA positive during follow up. No patient had minimal residual disease (MRD), within 1 month after surgery (+/- prior neoadjuvant systemic therapy). One case displayed cfDNA positive during surveillance (DDR2; c.2411T>C; p.F804S, TP53; c.743G>A; p.R248Q), at time-point of 7mos after surgery. The patient undergone mammogram, ultrasound and systemic work-up and yet, no evidence of recurrence was found till 6months after cfDNA positive conversion. Follow-up cfDNA analyses are necessary to evaluate mutation status whether, positive/increase VAF or negative conversion. At the same time, systemic radiology work-up needs to be done to investigate the development of clinically relevant recurrence after positive cfDNA, to confirm whether cfDNA positive finding was a false positive call or a matter of longer lead time. Anchor mutations selected from primary tumor was analyzed in serial cell-free DNA using mass spectrometry based UHS platform. With sufficient number of anchor mutations for tracking (>5), 100% sensitivity was observed with 0-10% false positivity. While liquid biopsy enable non-invasive surveillance, and yet needs substantial amount of blood draw 5-10ml each time, our platform using mass spectrometry can detect anchor mutation from less than 2ml whole blood. Clinical utility of cfDNA surveillance using UHS platform needs further evidence with longer follow-up analyses in larger prospective cohort. Citation Format: Jisun Kim, Whee Kyung Jo, Soo Jeong Choi, Hwan Park, Jiyoung Lee, Sae Byul Lee, Hee Jin Lee, Hee Jeong Kim, Il Yong Chung, Beom Seok Ko, Jong Won Lee, Byung Ho Son, Sei Hyun Ahn, Sung-Bae Kim, Kyung Hae Jung, Jin-Hee Ahn, Sung-Min Chun. Tracking anchor mutations in serial cell-free DNA using ultra-high sensitive mass spectrometry method provide risk of subsequent recurrence during surveillance after standard therapy [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 P5-01-11.

Alloseq HCT: A Wide Range Chimerism Testing Solution with High Sensitivity, Accuracy and Precision for Routine Post Hematopoietic Cell Transplant Monitoring

Genetic chimerism testing in hematopoietic cell transplant (HCT) recipients is typically done using methods based on Short Tandem Repeats (STR), with lower limit of detection around 2%. More sensitive chimerism detection methods may aid in engraftment monitoring and potentially predict early relapse of the disease. We have developed and analytically validated a highly sensitive chimerism test based on Next Generation Sequencing that utilizes single-nucleotide polymorphisms located throughout the genome. The test combines a clinical laboratory compatible protocol with streamlined workflow and a fully automated analysis software to accurately quantify the level of chimerism. Twenty-two samples designed to mimic genomic DNA (gDNA) derived from HCT recipient samples were generated using four distinct gDNA samples mixed at fractions ranging between 0.1-30%. These samples were used to validate the entire AlloSeq HCT™ workflow across multiple DNA input amounts, operators, reagent lots and sequencing runs. Sequencing was performed on the Illumina MiSeq and chimerism quantification was done using the AlloSeq HCT software. The data obtained by the AlloSeq HCT was compared to the expected mixtures and a correlation of 0.99 was obtained. The assay was sensitive down to 0.1%. We next evaluated within-run and across-run variability, which were on average 4.7% and 4.1%, respectively. The within-run and across-run variability was calculated over a range of input gDNA between 3 ng and 100 ng, showing the robustness of the method at different test conditions. AlloSeq HCT test accurately and reproducibly quantified the level of chimerism across a wide dynamic range with higher sensitivity than current standard of care. Combined with automated data analysis, 24-hour workflow and minimal hands-on time, AlloSeq HCT is an optimal method with high sensitivity and precision for routine chimerism testing.

Low-Pass Whole-Genome Sequencing as a Method of Determining Copy Number Variations in Uveal Melanoma Tissue Samples

Analysis of specific somatic copy number alterations (SCNAs) using multiplex ligation-dependent probe amplification (MLPA) is used routinely as a prognostic test for uveal melanoma (UM). This technique requires relatively large amounts of input DNA, unattainable from many small fine-needle aspirate biopsy specimens. Herein, we compared the use of MLPA with whole-genome amplification (WGA) combined with low-pass whole-genome sequencing (LP-WGS) for detection of SCNA profiles in UM biopsy specimens. DNA was extracted from 21 formalin-fixed, paraffin-embedded UM samples and SCNAs were assessed using MLPA and WGA followed by LP-WGS. Cohen's κ was used to assess the concordance of copy number calls of each individual chromosome arm for each patient. MLPA and WGA/LP-WGS detection of SCNAs in chromosomes 1p, 3, 6, and 8 were compared and found to be highly concordant with a Cohen's κ of 0.856 (bias-corrected and accelerated 95% CI, 0.770-0.934). Only 13 of 147 (8.8%) chromosomal arms investigated resulted in discordant calls, predominantly SCNAs detected by WGA/LP-WGS but not MLPA. These results indicate that LP-WGS might be a suitable alternative or adjunct to MLPA for the detection of SCNAs associated with prognosis of UM, for cases with limiting tissue or DNA yields.

Developmental validation of the monSTR identity panel, a forensic STR multiplex assay for massively parallel sequencing

The 21-plex STR panel monSTR was designed for high-fidelity forensic genotyping on the Illumina MiSeq platform. In this study, the panel’s performance was validated according to the recommended validation guidelines of the Scientific Working Group for DNA Analysis Methods (SWGDAM). Concordance, repeatability and reproducibility, sensitivity of detection, mixture analysis, species-specificity, and the ability to analyze mock samples were assessed. Sequence data was analyzed using the genotyping software toaSTR. The assay performance was evaluated by measuring the read on-target ratio, the genotype accuracy, the inter-locus balance, the heterozygosity balance, and the signal-to-noise ratio. Results showed that profiles of NIST reference DNA samples as well as GEDNAP proficiency samples were fully concordant with CE-based methods. In addition, inter-run and intra-run variation experiments indicated high precision. Furthermore, full profiles could be obtained using 62.5 pg of DNA input amount with proper inter-locus balance and read on-target ratio; 76.4% of alleles were correctly called with 7.8 pg DNA input amount. It was demonstrated that 94.4% of minor contributor alleles were resolved accurately in a 1:49 mixture. Results suggested that the minor contribution could be precisely calculated based on the minor component allele frequency. Validation results described here demonstrate that the monSTR forensic identity panel is a valid tool for forensic STR genotyping using massively parallel sequencing.

Microfluidic Platform for Next-Generation Sequencing Library Preparation with Low-Input Samples.

Advances in next-generation sequencing (NGS) have made available a wealth of information that had previously been inaccessible to researchers and clinicians. NGS has been applied to understand genomic, transcriptomic, and epigenomic changes and gained traction as a significant tool capable of accelerating diagnosis, prognosis, and biomarker discovery. However, these NGS assays have yet to be practical methods for patient stratification or diagnosis because of the gap between the tiny quantities of biomaterials provided by a clinical sample and the large DNA input required by most of these assays. Current library preparation methodologies typically require large input amounts of DNA and a long and complicated manual process. Here, we present a microfluidic droplet-based system for NGS library preparation, capable of reducing the number of pipetting steps significantly, reducing reagent consumption by 10×, andautomating much of the process, while supporting an extremely low DNA input requirement (10 pg per library). This semiautomated technology will allow for low-input preparations of 8 libraries simultaneously while reducing batch-to-batch variation and operator hands-on time.

Comparison of CE- and MPS-based analyses of forensic markers in a single cell after whole genome amplification

Whole genome amplification (WGA) allows for multiple genetic analyses with low template DNA, such as DNA derived from a single cell. WGA could increase the amount of input DNA from the pg to the μg level. However, there are no studies comparing the performance of forensic markers with DNA from a single cell after WGA evaluated on both capillary electrophoresis (CE) and massively parallel sequencing (MPS) platforms. In this study, cell lines consisting of female cultured B-lymphoblastoid cells and karyocytes from male venous blood were segregated into one, two, three and five cells. Including the references with the bulk cells, all samples were generated by WGA with the multiple displacement amplification (MDA) strategy in triplicate and genotyped on CE and MPS platforms. Allele balance, stutter ratio, accuracy, repeatability and concordance of short tandem repeat (STR) markers were used to evaluate the genotyping performance on both platforms. Additionally, the sequence coverage ratio (SCR) and SNP genotypes were evaluated for sequence information generated from the MPS. Heterozygous loci showed high allele balance, with an overall average allele balance ratio larger than 0.79 on the CE and 0.75 on the MPS platforms for the venous blood cell samples; the cultured B-lymphoblastoid cell samples had ratios of 0.62 and 0.70, respectively. The stutter ratio of every source and cell number from both cell line samples were very close, ranging from 5.3%–7.2% for autosomal STRs and approximately 10 % of Y chromosomal STRs on the CE platform. The average stutter, allele, and sequence-based and length-based noise ratios were 6.6 %, 88 %, 4.7 % and 0.7 %, respectively, in the single male cell sample. SNPs also showed high consistency and intralocus balance. Our study indicated that WGA with MDA strategy works relatively well of STR and SNP genotyping with low copy number samples on CE and MPS, even with one-cell sample.

Production of Lentiviral Vectors Using Suspension Cells Grown in Serum-free Media

Lentiviral vectors are increasingly utilized in cell and gene therapy applications because they efficiently transduce target cells such as hematopoietic stem cells and T cells. Large-scale production of current Good Manufacturing Practices-grade lentiviral vectors is limited because of the adherent, serum-dependent nature of HEK293T cells used in the manufacturing process. To optimize large-scale clinical-grade lentiviral vector production, we developed an improved production scheme by adapting HEK293T cells to grow in suspension using commercially available and chemically defined serum-free media. Lentiviral vectors with titers equivalent to those of HEK293T cells were produced from SJ293TS cells using optimized transfection conditions that reduced the required amount of plasmid DNA by 50%. Furthermore, purification of SJ293TS-derived lentiviral vectors at 1 L yielded a recovery of 55% ± 14% (n = 138) of transducing units in the starting material, more than a 2-fold increase over historical yields from adherent HEK293T serum-dependent lentiviral vector preparations. SJ293TS cells were stable to produce lentiviral vectors over 4 months of continuous culture. SJ293TS-derived lentiviral vectors efficiently transduced primary hematopoietic stem cells and T cells from healthy donors. Overall, our SJ293TS cell line enables high-titer vector production in serum-free conditions while reducing the amount of input DNA required, resulting in a highly efficient manufacturing option.

Comprehensive Evaluation and Validation of a Next-Generation Sequencing Assay for Minimal Residual Disease Detection in T-Lymphoblastic Leukemia/Lymphoma

Introduction: Detection of minimal residual disease (MRD) in T-lymphoblastic leukemia/lymphoma (T-ALL) has been used in post-therapy monitoring and outcome prediction. Currently, multi-color flow cytometry is widely used for MRD detection with limit of detection (LOD) of 0.1 to 0.01% depending on the clinical implementation. However, flow cytometry assays require extensive training and experience for accurate interpretation and standardization is challenging. Next generation sequencing (NGS) assays with panels targeting the inherent polymorphism of the immunoglobulin genes have been applied for MRD detection in B cell neoplasms, including an assay for myeloma and B-ALL MRD detection developed by Adaptive Biotechnologies (Seattle, WA) that was granted FDA approval for clinical testing. We have published a similar approach for T-ALL MRD detection using the polymorphic T-cell receptor (TCR) genes. These assays offer higher sensitivity, easier standardization, and more objective interpretation. Here we present our comprehensive evaluation and validation of a T-ALL MRD assay using the Adaptive Biotechnologies immunoSEQ kit for potential clinical application. Methods: NGS was performed using the Adaptive Biotechnologies immunoSeq kit and illumina NextSeq 500 according to manufacturer's instruction. Quantification of T cell repertoire was performed using the Adaptive Biotechnologies analysis pipeline. A single TCRβ sequence with ≥19% frequency of the T-cell population in diagnostic samples was defined as a T-ALL clone. Positive MRD was defined as detection of one or more such T-ALL sequences having a 100% DNA sequence match in the corresponding MRD samples. T-ALL leukemic cells and normal T cells were sorted from 2 bone marrow aspirates and peripheral blood from 20 healthy donors, respectively. T-ALL cells were spiked into pooled normal T cells to create a series of diagnostic T-ALL standards and MRD T-ALL standards down to 0.001%. Prior to assaying the standards, a T-ALL specimen was assessed at 11 different DNA input levels ranging from 3-1000ng, and interference of non-T-cell DNA was tested. For all other assays, 800ng DNA was used in two independent hsTCRB replicates for T-ALL clone identification in diagnostic samples and 2,400ng DNA was used in six independent hsTCRB replicates for T-ALL clone detection in MRD samples. Within run precision was assessed by assaying all standards and 3 pairs of diagnostic and MRD samples in triplicate. Between run precision was assessed by assaying 1 standard and 4 samples on two different days by the same technologist. Intermediate precision was assessed by assaying 3 pairs of diagnostic and MRD samples by two different technologists. LOD was determined as the lowest frequency at which MRD could be detected in MRD standards and Limit of Quantitation (LOQ) determined as the level below which the coefficient of variation (CV) >25% in MRD standards. Results: The %CV for T-ALL quantification across the range of DNA input was 4.5%, suggesting independence of quantification from DNA input amount. Non-T-cell DNA has almost no impact on T-ALL quantification. T-ALL clones were detected with 100% concordance in diagnostic and MRD standards. The %CV of within and between run precision and intermediate precision were < 25% in T-ALL identification assay and all MRD detection assays except at 0.001% MRD, in which %CV was 43.8%. Quantification of T-ALL in both diagnostic and MRD standards were highly correlated with expected values (R2 = 0.96, slope = 0.89 and R2 = 1.00, slope = 0.88, respectively). T-ALL identification and MRD detection in specimens stored at 4°C or at room temperature for up to 14 days were within 25% CV as compared to fresh specimens. The assay was then performed on 40 diagnostic clinical T-ALL samples and T-ALL clonal sequences were identified in 34 samples (85%). Conclusions: The immunoSEQ assay is evaluated and technically validated for clinical application, but requires validation in a larger cohort of clinical samples with concurrent outcome data. The assay is suitable for roughly 70% T-ALL where TCRβ is rearranged. The assay LOD for T-ALL MRD detection is 0.001% and LOQ is 0.01%, offering the possibility of improved sensitivity and more objective interpretation. Disclosures No relevant conflicts of interest to declare.

Comprehensive DNA 5-Hydroxymethylation Landscapes in Myelodysplastic Syndromes (MDS)

Background: Epigenetic regulators as well as epigenetic marks play an essential role in regulating normal hematopoiesis. Epigenetic alterations are one of the early events reflecting changes in cell identify during the transition of a pre-leukemic condition into a malignant phenotype. Capturing epigenetic alterations at early stages of pre-leukemic conditions could benefit the diagnosis and understanding the disease progression during hematopoietic malignant transformation. DNA 5-hydroxymethylation (5hmC) is the one of the major oxidation products of 5-methycytosine (5mC) mediated by the TET protein family dioxygenase. 5hmC has been reported to be involve in the regulation of chromatin accessibility and gene transcription. The dynamic changes of 5hmC have been reported as a hallmark in myeloid malignancies. In our study, we applied an improved 5hmC profiling method (sCMSIP) developed in our lab to systematically profile 5hmC and evaluate the 5hmC dynamics in patients with myeloid malignancies. Methods: In order to profile 5hmC using low amount of input DNA isolated from patient bone-marrow aspirates, we improved our previously developed anti-CMSIP method to capture 5hmC enriched regions using ultra-low amount of genomic DNA (10-20 ng gDNA). Cytosine-5-methylenesulfonatem (CMS) is the 5hmC derivatives upon sodium bisulfite treatment. A home-made anti-CMS antibody was developed to specifically recognize CMS genome-wide after sodium bisulfite treatment. The traditional anti-CMSIP has been widely used to study the genome-wide 5hmC distribution in various biological systems, including mESC, HSC and immune cells. We further optimized the immunoprecipitation procedure to profile genome-wide 5hmC with ultra-low amount of genomic DNA (sensitive CMSIP, sCMSIP). Using this improved CMS-IP method, we performed genome-wide 5hmC analysis in ~100 individuals including healthy donors (n = 10), patients with MDS (n = 62), AML (n = 4) and CMML (n = 13). In parallel, we performed RRBS (DNA methylation) and RNA-seq (transcriptome) analysis on matched samples. We then performed integrative bioinformatics analyses to unveil the potential diagnostic and prognostic values of 5hmC in myeloid neoplasms. Results and Conclusions: Based on our analysis, we found distinct epigenetic landscapes and transcriptomes between healthy donor and patients with myeloid malignancies. Interestingly, we could further separate patients with MDS into three clusters by comparing their 5hmC landscapes with normal, AML and CMML individuals, suggesting that 5hmC might be a sensitive epigenetic mark to reflect the disease status of MDS. Furthermore, we identified a positive correlation between 5hmC distribution and the blast ratio in paired patients before and after hypomethylating agent (HMA) treatment. These findings suggest that 5hmC might be used as a prognostic marker for HMA treatment. Detailed analyses further elucidate the impaired 5hmC enrichment in various key transcription factors that are essential for HSC function and myeloid lineage specification. Disclosures Garcia-Manero: Amphivena: Consultancy, Research Funding; Helsinn: Research Funding; Novartis: Research Funding; AbbVie: Research Funding; Celgene: Consultancy, Research Funding; Astex: Consultancy, Research Funding; Onconova: Research Funding; H3 Biomedicine: Research Funding; Merck: Research Funding.

Phased Variant Enrichment for Enhanced Minimal Residual Disease Detection from Cell-Free DNA

Background: Circulating tumor DNA (ctDNA) is an emerging biomarker in non-Hodgkin lymphomas (NHLs). Current methods for ctDNA minimal residual disease (MRD) are limited by two factors - low input DNA amounts and high background error rates. VDJ sequencing (i.e., IgHTS) has low background but is limited by low cell-free DNA (cfDNA). Tracking multiple mutations via CAPP-Seq improves sensitivity, but detection is limited by background errors. Clustered mutations have been described in multiple cancers including NHLs and potentially have lower error rates. We explored clustered mutations from whole-genome sequencing (WGS) to identify 'phased variants' (PVs), defined as multiple mutations on a single DNA molecule (Fig 1A). We designed a method to capture PVs for improved ctDNA detection and explored its utility for MRD in DLBCL. Methods: We reanalyzed WGS from 1455 tumors across 11 cancer types. We identified genomic regions recurrently containing PVs and designed an assay for deep cfDNA sequencing. We applied this assay to 171 patients with large B-cell lymphomas. We compared the performance of PVs for disease detection to current ctDNA techniques, including CAPP-Seq and duplex sequencing. Results: To utilize PVs, mutations must occur within a typical cfDNA strand (~170bp). We measured the frequency of putative PVs in WGS, focusing on pairs of mutations occurring within <170bp. PVs were more frequent in NHLs than any other histology (median: DLBCL, 642; FL, 307; Burkitt, 89.5; CLL, 34; breast, 46; lung, colorectal, melanoma, bladder, cervical, head & neck < 10 per case; P < 0.001 for NHLs vs others). PVs in NHLs were enriched in single base substitution mutational signatures associated with activation-induced cytidine deaminase (AID) (SBS84 & 85). PVs in NHLs occurred in stereotyped regions, including canonical AID targets such as IGH, IGK, and IGL, as well as 44 other AID targets (Schmitz, NEJM 2018) (Fig 1B). We additionally identified novel regions not previously implicated as targets of AID, including LPP, XBP1, BZRAP1, and HLA-DQ. We designed an approach for enriching PVs from ~115kb (Phased variant Enrichment Sequencing, PhasE-Seq) and other regions recurrently mutated in B-NHLs. We compared PhasE-Seq and CAPP-Seq using tumor and plasma samples from 16 patients. Compared to CAPP-Seq, PhasE-Seq yielded more SNVs and PVs per case (median SNVs: 331 vs 114, P<0.001; PVs: 729 vs 222.5, P <0.001). We next applied PhasE-Seq to 171 patients with untreated lymphomas (DLBCL, 148; primary mediastinal B-cell lymphoma, PMBCL, 23) profiling 58 tumor and 171 plasma samples with matched germline. We observed significant differences in the distribution of PVs between subtypes - for example, GCB-DLBCL had more PVs in BCL2, MYC, and SGK1, while ABC-DLBCL had more in PIM1 and IGHV4-34 (Fig 1C). Similarly, we noted enrichment in PVs in PMBCL in CIITA, SOCS1, CD83, and ITPKB. We then compared PhasE-Seq to alternative methods for MRD detection. We used limiting dilutions of patient ctDNA down to 1:1,000,000 to establish the detection limit (LOD, Fig 1D). PhasE-Seq outperformed CAPP-Seq and duplex sequencing for recovery of expected tumor content, with a high degree of linearity down to ~1:1,000,000. We applied standard CAPP-Seq and PhasE-Seq to patient cfDNA samples after two cycles of front-line therapy (n=92). We previously reported a 2.5-log reduction in ctDNA as prognostic at this time-point (Kurtz, JCO 2018). Using CAPP-Seq, 58% (53/92) of samples were undetectable. Using PhasE-Seq, 30% (16/53) of samples not detected by CAPP-Seq had evidence of MRD, with levels as low as 2:1,000,000. In patients with ctDNA undetected by CAPP-Seq, detection by PhasE-Seq significantly stratified outcomes (Fig 1E). Conclusions: PVs are frequent in NHLs, likely due to AID, and correlate with disease biology. PhasE-Seq allows for superior detection of ctDNA, including MRD detection in the majority of patients after 2 cycles. Targeted sequencing of ctDNA should consider PVs to maximize detection and guide precision approaches. Figure 1: A) Structure of phased variants B) Distribution of putative PVs from WGS data C) Genomic enrichment in PVs in lymphoma subtypes D) Dilution series comparing PhasE-Seq, CAPP-Seq, and duplex sequencing E) Waterfall plot showing ctDNA level vs outcome; undetectable ctDNA by CAPP-Seq is highlighted F) EFS of patients with undetectable ctDNA by CAPP-Seq after 2 cycles, stratified by PhasE-Seq Disclosures Kurtz: Roche: Consultancy. Diehn:Roche: Consultancy; AstraZeneca: Consultancy; Novartis: Consultancy; BioNTech: Consultancy; Quanticell: Consultancy. Alizadeh:Pharmacyclics: Consultancy; Janssen: Consultancy; Genentech: Consultancy; Roche: Consultancy; Gilead: Consultancy; Celgene: Consultancy; Chugai: Consultancy; Pfizer: Research Funding.

Ultralow amounts of DNA from long-term archived serum samples produce quality genotypes

While genotyping studies are scavenging for suitable samples to analyze, large serum collections are currently left unused as they are assumed to provide insufficient amounts of DNA for array-based genotyping. Long-term stored serum is considered to be difficult to genotype since preanalytical treatments and storage effects on DNA yields are not well understood. Successful genotyping of such samples has the potential to activate large biobanks for future genome-wide association studies (GWAS). We aimed to evaluate genotyping of ultralow amounts of DNA from samples stored up to 45 years in the Janus Serum Bank with two commercially available platforms. 64 samples, with various preanalytical treatments, were genotyped on the Axiom Array from Thermo Fisher Scientific and a subset of 24 samples with slightly higher yield were genotyped on the HumanCoreExome array from Illumina. Our results showed that about 80% of the serum samples produced call rates with the Axiom arrays that would be satisfactory in GWAS. The mean DNA yield was 5.8 ng as measured with PicoGreen, 3–6% of recommended yield. The failed samples had on average lower input amounts of DNA. All serum samples genotyped on the HumanCoreExome with a standard and FFPE protocol produced GWAS satisfactory call rates, with mean 97.57% and 98.35% call rates, respectively. The mean yield was 10.65 ng, 6% of the recommendations. Successful array-based genotyping of ultralow DNA yields from serum samples stored up to 45 years is possible. These results demonstrate the potential to activate large serum biobank collections for future studies.