Double-stranded Library Preparation Research Articles

Abstract 960: Improved cfDNA methylation profiling through correction of misrepaired jagged-ends

Abstract Background: Profiling cfDNA methylation enables early cancer detection and classification, but challenges arise from "jagged ends" (JEs) for ~90% of cfDNA molecules, as well as for FFPE samples and partially degraded genomic DNA. Repair of these jagged ends introduces erroneous methylation signals, especially in shorter cfDNA fragments enriched for tumor derived molecules. Separately, DNA conversions during 5mC profiling hamper accurate simultaneous ctDNA genotyping of somatic mutations. To meet these needs, we developed a method preserving double-stranded cfDNA molecules without error-prone end-repair at jagged ends. This enables superior methylation profiling including hemimethylation detection, and simultaneous ctDNA SNV genotyping. Methods: We present JEEPERS (Jagged-End Error Polishing of Enzymatically misRepaired Sequences), a novel method for correcting errors in double-stranded library preparation for methylation profiling. JEEPERS detects and quantifies enzymatic repair errors at JEs, correcting them in silico. Leveraging duplex UMIs, it utilizes complementary strands and sibling reads from cfDNA families without jagged ends. The method exploits the 5' to 3' polarity of ER-associated errors and JE length profiles for correction. JEEPERS introduces anchor CpGs for precise correction of rare signals and enables accurate SNV calling at non CpG sites, addressing the challenge of differentiating true SNVs from conversion artifacts leading to C>T/G>A changes in sequencing data. Results: Analyzing 248 cfDNA samples (139 NSCLC patients, 109 healthy controls) by methylation profiling (EM-Seq) and ctDNA mutation genotyping (CAPP-Seq), JEEPERS correction efficiently addressed distortions, even at low tumor concentrations. Limiting dilution experiments demonstrated JEEPERS achieving a LOD95 of ~0.2% with 95% specificity (n=300). Despite ~50% lower molecular depth, JEEPERS-corrected cfDNA EM-Seq enabled accurate simultaneous methylation profiling and ctDNA genotyping, correlating strongly with CAPP-Seq genotypes (R=0.998, n=30). As expected, mutant ctDNA molecules were shorter than wild-type in both CAPP-Seq and EM-Seq data. cfDNA methylation patterns varied by oncogene vs. tumor suppressor status; EGFR mutants showed increased methylation (p<0.01), while TP53 mutants had lower methylation levels (p<0.05). Conclusions: Our method effectively corrects cfDNA methylation data, even at low tumor concentration, ensuring enhanced methylation signal accuracy and proficient SNV calling capabilities. This integrated approach offers a powerful means for investigating the link between SNVs and DNA methylation, significantly enhancing the accuracy of cancer detection using cfDNA, especially for early-stage tumors. This paves the way for a deeper understanding of the molecular mechanisms driving cancer initiation and progression. Citation Format: Rui Wang, Emily G. Hamilton, Diego Almanza, Angela Hui, Maximilian Diehn, Ash A. Alizadeh. Improved cfDNA methylation profiling through correction of misrepaired jagged-ends [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 960.

Investigation of Different Library Preparation and Tissue of Origin Deconvolution Methods for Urine and Plasma cfDNA Methylome Analysis.

Methylation sequencing is a promising approach to infer the tissue of origin of cell-free DNA (cfDNA). In this study, a single- and a double-stranded library preparation approach were evaluated with respect to their technical biases when applied on cfDNA from plasma and urine. Additionally, tissue of origin (TOO) proportions were evaluated using two deconvolution methods. Sequencing cfDNA from urine using the double-stranded method resulted in a substantial within-read methylation bias and a lower global methylation (56.0% vs. 75.8%, p ≤ 0.0001) compared to plasma cfDNA, both of which were not observed with the single-stranded approach. Individual CpG site-based TOO deconvolution resulted in a significantly increased proportion of undetermined TOO with the double-stranded method (urine: 32.3% vs. 1.9%; plasma: 5.9% vs. 0.04%; p ≤ 0.0001), but no major differences in proportions of individual cell types. In contrast, fragment-level deconvolution led to multiple cell types, with significantly different TOO proportions between the two methods. This study thus outlines potential limitations of double-stranded library preparation for methylation analysis of cfDNA especially for urinary cfDNA. While the double-stranded method allows jagged end analysis in addition to TOO analysis, it leads to significant methylation bias in urinary cfDNA, which single-stranded methods can overcome.

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.

Ancient DNA Methods Improve Forensic DNA Profiling of Korean War and World War II Unknowns.

The integration of massively parallel sequencing (MPS) technology into forensic casework has been of particular benefit to the identification of unknown military service members. However, highly degraded or chemically treated skeletal remains often fail to provide usable DNA profiles, even with sensitive mitochondrial (mt) DNA capture and MPS methods. In parallel, the ancient DNA field has developed workflows specifically for degraded DNA, resulting in the successful recovery of nuclear DNA and mtDNA from skeletal remains as well as sediment over 100,000 years old. In this study we use a set of disinterred skeletal remains from the Korean War and World War II to test if ancient DNA extraction and library preparation methods improve forensic DNA profiling. We identified an ancient DNA extraction protocol that resulted in the recovery of significantly more human mtDNA fragments than protocols previously used in casework. In addition, utilizing single-stranded rather than double-stranded library preparation resulted in increased attainment of reportable mtDNA profiles. This study emphasizes that the combination of ancient DNA extraction and library preparation methods evaluated here increases the success rate of DNA profiling, and likelihood of identifying historical remains.

Testing a series of modifications on genomic library preparation methods for ancient or degraded DNA

Technological advancements have revolutionized ancient and degraded DNA analysis, moving the field to the Next Generation Sequencing era. One of the advancements, the ancient DNA-oriented high-throughput library preparation methods, enabled the sequencing of more endogenous molecules. Although fairly optimized, both single- and double-stranded library preparation methods hold the potential for further improvement. Here, we test a series of modifications made at different steps of both single- and double-stranded library preparation methods. Given all the modifications tested, we found that two of them provide further benefits, including the use of Endonuclease VIII as a pre-treatment step before preparing single-stranded libraries and the use of a modified second adapter of the single stranded-libraries as an alternative option to enable sequencing of single stranded-libraries with the standard Illumina sequencing primer instead of the custom designed as described in the single stranded library preparation method. Furthermore, we propose uracil-DNA-glycosylase (UDG) could also be considered for both single- and double-stranded library preparation methods, although additional parameters should be taken into account depending on the sequencing strategy and the sample characteristics. Further modifications were also tested and although they were not advantageous, they could be considered as equivalent to the published options.

Cost-effective straightforward method for captured whole mitogenome sequencing of ancient DNA

Working with mitochondrial DNA from highly degraded samples is challenging. We present a whole mitogenome Illumina-based sequencing method suitable for highly degraded samples. The method makes use of double-stranded library preparation with hybridization-based target enrichment. The aim of the study was to implement a new user-friendly method for analysing many ancient DNA samples at low cost. The method combines the Swift 2S™ Turbo library preparation kit and xGen® panel for mitogenome enrichment. Swift allows to use low input of aDNA and own adapters and primers, handles inhibitors well, and has only two purification steps. xGen is straightforward to use and is able to leverage already pooled libraries. Given the ancient DNA is more challenging to work with, the protocol was developed with several improvements, especially multiplying DNA input in case of low concentration DNA extractions followed by AMPure® beads size selection and real-time pre-capture PCR monitoring in order to avoid cycle-optimization step.Nine out of eleven analysed samples successfully retrieved mitogenomes. Hence, our method provides an effective analysis of whole mtDNA, and has proven to be fast, cost-effective, straightforward, with utilisation in population-wide research of burial sites.

Double-Stranded Library Preparation for Ancient and Other Degraded Samples.

High-throughput sequencing (HTS) allows fast and cost-efficient sequencing of ancient DNA (aDNA) without prior information about what sequences should be targeted. One necessary step for HTS is the preparation of a sequencing library. Commercial kits are available for this purpose, but many of these are not suitable for aDNA or other types of damaged DNA. Here, we outline a protocol for HTS library preparation that is optimized for ancient DNA. We report the library conversion rate for a range of input template and adapter concentrations. Our results show that the protocol performs at a high efficiency.

Experimental approaches for ancient DNA extraction and sample preparation for next generation sequencing in ultra-clean conditions

In this study, we aimed to develop an approach for genetic material extraction and sample preparation of ancient DNA for next generation sequencing that minimizes intra-laboratory contamination. A special module system consisting of four sterile gloveboxes connected by antechambers, which maintain a high purity atmosphere and allow conducting all stages of sample preparation in an isolated clean environment, starting with the processing of archaeological material, was developed. Furthermore, a fast and cost-effective double-stranded library preparation protocol for massive parallel sequencing was developed using non-standard modified sequencing adaptors, which enabled obtaining a library of fragments from extremely low amounts of starting degraded material. Bioinformatics data processing showed that the distribution of substitution frequencies in a subset of reads, mapped to the human reference genome, completely coincides with the pattern of postmortem modifications expected when the double-stranded library preparation method is employed. This is a strong evidence for the endogenous origin of the sequenced fragments of the human genome. Thus, the results of ancient sample and negative control sample sequencing, indicated the absence of visible contamination during sample preparation procedure.