Amplification-free Library Preparation Research Articles

A Two-color Single-molecule Sequencing Platform and Its Clinical Applications.

DNA sequencers have become increasingly important research and diagnostic tools over the past 20 years. In this study, we developed a single-molecule desktop sequencer, GenoCare 1600 (GenoCare), which utilizes amplification-free library preparation and two-color sequencing-by-synthesis chemistry, making it more user-friendly compared with previous single-molecule sequencing platforms for clinical use. Using the GenoCare platform, we sequenced an Escherichia coli standard sample and achieved a consensus accuracy exceeding 99.99%. We also evaluated the sequencing performance of this platform in microbial mixtures and coronavirus disease 2019 (COVID-19) samples from throat swabs. Our findings indicate that the GenoCare platform allows for microbial quantitation, sensitive identification of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, and accurate detection of virus mutations, as confirmed by Sanger sequencing, demonstrating its remarkable potential in clinical application.

A highly efficient scheme for library preparation from single-stranded DNA

Although methods for sequencing library preparation from double-stranded DNA are well established, those from single-stranded DNA (ssDNA) have not been well studied. Further, the existing methods have limitations in efficiency and yield. Therefore, we developed a highly efficient procedure for sequencing library preparation from ssDNA. In this method, the first adaptor tagging of ssDNA is performed using terminal deoxyribonucleotidyl transferase (TdT)-assisted adenylate connector-mediated ssDNA (TACS) ligation, which we reported recently. After complementary strand synthesis using the adaptor-tagged ssDNA, second adaptor tagging via Vaccinia virus topoisomerase I (VTopoI or TOPO)-based adaptor ligation is performed. With additional steps for degradation, repression, and removal of the adaptor dimer, the proposed TACS-TOPO scheme realizes adaptor dimer-free sequencing library preparation from ssDNA samples of 24 pg. The TACS-TOPO scheme was successfully applied to cell-free DNA analysis with amplification-free library preparation from 50 µL of human serum. A modified TACS-TOPO scheme was also applied to DNA extracted from ancient human bones, bringing two to eight times more library yields than those using a conventional library preparation protocol. The procedures for preparing VTopoI and its complex with a double-stranded oligonucleotide adaptor are also described. Overall, the proposed TACS-TOPO scheme can facilitate practical and sensitive sequencing analysis of ssDNA.

Targeted DNA methylation analysis and prediction of smoking habits in blood based on massively parallel sequencing

Tobacco smoking is a frequent habit sustained by > 1.3 billion people in 2020 and the leading preventable factor for health risk and premature mortality worldwide. In the forensic context, predicting smoking habits from biological samples may allow broadening DNA phenotyping. In this study, we aimed to implement previously published smoking habit classification models based on blood DNA methylation at 13 CpGs. First, we developed a matching lab tool based on bisulfite conversion and multiplex PCR followed by amplification-free library preparation and targeted paired-end massively parallel sequencing (MPS). Analysis of six technical duplicates revealed high reproducibility of methylation measurements (Pearson correlation of 0.983). Artificially methylated standards uncovered marker-specific amplification bias, which we corrected via bi-exponential models. We then applied our MPS tool to 232 blood samples from Europeans of a wide age range, of which 90 were current, 71 former and 71 never smokers. On average, we obtained 189,000 reads/sample and 15,000 reads/CpG, without marker drop-out. Methylation distributions per smoking category roughly corresponded to previous microarray analysis, showcasing large inter-individual variation but with technology-driven bias. Methylation at 11 out of 13 smoking-CpGs correlated with daily cigarettes in current smokers, while solely one was weakly correlated with time since cessation in former smokers. Interestingly, eight smoking-CpGs correlated with age, and one displayed weak but significant sex-associated methylation differences. Using bias-uncorrected MPS data, smoking habits were relatively accurately predicted using both two- (current/non-current) and three- (never/former/current) category model, but bias correction resulted in worse prediction performance for both models. Finally, to account for technology-driven variation, we built new, joint models with inter-technology corrections, which resulted in improved prediction results for both models, with or without PCR bias correction (e.g. MPS cross-validation F1-score > 0.8; 2-categories). Overall, our novel assay takes us one step closer towards the forensic application of viable smoking habit prediction from blood traces. However, future research is needed towards forensically validating the assay, especially in terms of sensitivity. We also need to further shed light on the employed biomarkers, particularly on the mechanistics, tissue specificity and putative confounders of smoking epigenetic signatures.

Amplification-free library preparation with SAFE Hi-C uses ligation products for deep sequencing to improve traditional Hi-C analysis

PCR amplification of Hi-C libraries introduces unusable duplicates and results in a biased representation of chromatin interactions. We present a simplified, fast, and economically efficient Hi-C library preparation procedure, SAFE Hi-C, which generates sufficient non-amplified ligation products for deep sequencing from 30 million Drosophila cells. Comprehensive analysis of the resulting data shows that amplification-free Hi-C preserves higher complexity of chromatin interaction and lowers sequencing depth for the same number of unique paired reads. For human cells which have a large genome, SAFE Hi-C recovers enough ligated fragments for direct high-throughput sequencing without amplification from as few as 250,000 cells. Comparison with published in situ Hi-C data from millions of human cells demonstrates that amplification introduces distance-dependent amplification bias, which results in an increased background noise level against genomic distance. With amplification bias avoided, SAFE Hi-C may produce a chromatin interaction network more faithfully reflecting the real three-dimensional genomic architecture.

Short tandem repeat stutter model inferred from direct measurement of in vitro stutter noise.

Short tandem repeats (STRs) are polymorphic genomic loci valuable for various applications such as research, diagnostics and forensics. However, their polymorphic nature also introduces noise during in vitro amplification, making them difficult to analyze. Although it is possible to overcome stutter noise by using amplification-free library preparation, such protocols are presently incompatible with single cell analysis and with targeted-enrichment protocols. To address this challenge, we have designed a method for direct measurement of in vitro noise. Using a synthetic STR sequencing library, we have calibrated a Markov model for the prediction of stutter patterns at any amplification cycle. By employing this model, we have managed to genotype accurately cases of severe amplification bias, and biallelic STR signals, and validated our model for several high-fidelity PCR enzymes. Finally, we compared this model in the context of a naïve STR genotyping strategy against the state-of-the-art on a benchmark of single cells, demonstrating superior accuracy.

Using Whole Genome Sequencing (WGS) of 3256 Patients with Hematologic Malignancies to Determine Genome Instability

Background:Genome instability is a hallmark of cancer. Mutations in DNA repair pathway genes are frequent in a number of solid tumors. Defects in DNA repair or damage response can weaken response to conventional chemotherapy and are frequently regarded as poor prognostic markers. However, a high tumor mutation burden (TMB, number of somatic mutations per mega base) was recently found to correlate with better response to immune checkpoint inhibitors e.g. in colon cancer. Patients with defects in the DNA mismatch repair (MMR) pathway in solid tumors are among the cases with the highest TMB. Hematological malignancies are generally expected on the lower end of the TMB spectrum. We used whole genome sequencing (WGS) for 3256 patients with hematological malignancies (lymphatic and myeloid) to determine factors of genetic instability across all entities.Aim:To determine the number of known mutations in genes from the DNA repair pathwayTo estimate TMB using WGS and identify cases with high TMB in hematologic malignanciesMethods:We investigated a cohort of 3256 patients with hematological malignancies, who were analyzed according to WHO diagnostic gold standards for routine purposes (incl. 584 acute myeloid leukemia [AML] and 635 myelodysplastic syndromes [MDS] samples). We performed amplification-free library preparation and sequencing on HiseqX and NovaSeq 6000 with a median coverage of 106x. Mapping and variant calling was performed with standard pipelines via BaseSpace (all Illumina, San Diego, CA). A pool of gender-matched genomic DNA (Promega, Madison, WI) was used for a tumor-unmatched normal variant calling. (a) In detail we evaluated 180 genes involved in DNA repair. We filtered on (likely) pathogenic variants from ClinVar and for TP53 on protein-truncating variants and (likely, possibly) pathogenic variants from the IARC database. (b) For TBM calculation we determined protein-altering changes and then subtracted all gnomAD listed variants in order to eliminate most germline variants.Results:We found 479 of 3256 (15%) patients with at least one pathogenic variant according to current database annotations in DNA repair or damage response genes. Most pathogenic variants were found in TP53 (330/3256; 10%) and ATM (25/3256, 1%), however, this is probably the effect of the already available systematic database annotation for both genes. For routine diagnostic purposes TP53 mutation status had been analyzed for 1184 patients with Sanger sequencing (7%) or amplicon next-generation sequencing (93%). A 98% and 99% concordance of the pathogenic and non-pathogenic TP53 status was found in comparison to WGS. Mutations in genes from the DNA double-strand break repair (and/or homologous DNA pairing and strand exchange) pathway were found in 93 patients (3%). Pathogenic and potentially germline MMR gene mutations were found in only 3 patients (0.1%, 2 MLH1, 1 MSH6), which equals the expected frequency in the Western population (0.05-0.3%).Next, we calculated TMB. The average was 2.4 [range: 0.4-39.2]. Only samples above the 95th percentile were defined as “TMBhi” (TMB ≥5). TMB was lowest in chronic myeloid leukemia (CML) and essential thrombocythemia (ET) (< 2) and no ET or CML patient was found among the TMBhi. We then focused on MDS, which is our largest subcohort: 56 of 635 (9%) patients were in TMBhi. Furthermore, among MDS patients, a significantly higher TMB was found in MDS-EB-2 (average 3.3 vs. 2.3 for non EB-2, p<.001) and a significantly lower TMB in MDS with isolated del(5q) (1.7 vs. 2.6 in all other MDS, p<.001).There was no association of high TMB and annotated DNA repair gene mutations. The three MMR deficient patients had an average TMB (1.8-2.2) in the hematologic sample. However, TMB was positively correlated with age (Spearman's rank correlation, p<.001) and average TMB was lower in patients under 60 years (2.3 vs. 2.5, p=0.007, t-test).Conclusions:High TMB is rare in hematological neoplasms, and our data across all entities suggest that the acquisition of mutations over age should be a major contributing factor.While TP53 and a few other factors are well studied, using genome-wide datasets in the near future will allow to deeply understand the broad patterns or signatures of genome stability (incl. copy number or structural variants) and their prognostic or predictive value. DisclosuresBaer:MLL Munich Leukemia Laboratory: Employment. Hutter:MLL Munich Leukemia Laboratory: Employment. Höllein:MLL Munich Leukemia Laboratory: Employment. Walter:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Comparison of whole-genome bisulfite sequencing library preparation strategies identifies sources of biases affecting DNA methylation data

BackgroundWhole-genome bisulfite sequencing (WGBS) is becoming an increasingly accessible technique, used widely for both fundamental and disease-oriented research. Library preparation methods benefit from a variety of available kits, polymerases and bisulfite conversion protocols. Although some steps in the procedure, such as PCR amplification, are known to introduce biases, a systematic evaluation of biases in WGBS strategies is missing.ResultsWe perform a comparative analysis of several commonly used pre- and post-bisulfite WGBS library preparation protocols for their performance and quality of sequencing outputs. Our results show that bisulfite conversion per se is the main trigger of pronounced sequencing biases, and PCR amplification builds on these underlying artefacts. The majority of standard library preparation methods yield a significantly biased sequence output and overestimate global methylation. Importantly, both absolute and relative methylation levels at specific genomic regions vary substantially between methods, with clear implications for DNA methylation studies.ConclusionsWe show that amplification-free library preparation is the least biased approach for WGBS. In protocols with amplification, the choice of bisulfite conversion protocol or polymerase can significantly minimize artefacts. To aid with the quality assessment of existing WGBS datasets, we have integrated a bias diagnostic tool in the Bismark package and offer several approaches for consideration during the preparation and analysis of WGBS datasets.

CRISPR\u2013Cas9-targeted fragmentation and selective sequencing enable massively parallel microsatellite analysis

Microsatellites are multi-allelic and composed of short tandem repeats (STRs) with individual motifs composed of mononucleotides, dinucleotides or higher including hexamers. Next-generation sequencing approaches and other STR assays rely on a limited number of PCR amplicons, typically in the tens. Here, we demonstrate STR-Seq, a next-generation sequencing technology that analyses over 2,000 STRs in parallel, and provides the accurate genotyping of microsatellites. STR-Seq employs in vitro CRISPR–Cas9-targeted fragmentation to produce specific DNA molecules covering the complete microsatellite sequence. Amplification-free library preparation provides single molecule sequences without unique molecular barcodes. STR-selective primers enable massively parallel, targeted sequencing of large STR sets. Overall, STR-Seq has higher throughput, improved accuracy and provides a greater number of informative haplotypes compared with other microsatellite analysis approaches. With these new features, STR-Seq can identify a 0.1% minor genome fraction in a DNA mixture composed of different, unrelated samples.

Microsatellite Marker Discovery using Single Molecule Real-Time Circular Consensus Sequencing on the Pacific Biosciences RS

Microsatellite sequences are important markers for population genetics studies. In the past, the development of adequate microsatellite primers has been cumbersome. However with the advent of next-generation sequencing technologies, marker identification in genomes of non-model species has been greatly simplified. Here we describe microsatellite discovery on a Pacific Biosciences single molecule real-time sequencer. For the Greater White-fronted Goose (Anser albifrons), we identified 316 microsatellite loci in a single genome shotgun sequencing experiment. We found that the capability of handling large insert sizes and high quality circular consensus sequences provides an advantage over short read technologies for primer design. Combined with a straightforward amplification-free library preparation, PacBio sequencing is an economically viable alternative for microsatellite discovery and subsequent PCR primer design.

Optimal enzymes for amplifying sequencing libraries

PCR amplification introduces bias into Illumina sequencing libraries1. Although amplification-free library preparation solves this, micrograms of starting material are usually required. Most researchers follow standard protocols using Phusion polymerase, which has processivity and fidelity advantages over most polymerases. Yet for genomics applications, our demands on DNA amplification systems often surpass their specification. Thermostable DNA polymerases such as Phusion are used to amplify mixtures of fragments, albeit with variable efficiency. Typically, (G+C)-neutral fragments are amplified with higher efficiency than extremely (G+C)-rich or (A+T)-rich fragments. The accumulation of these slight differences in amplification over multiple cycles often results in profound bias. There have been reports of using alternative DNA polymerases for Illumina library construction2, 3, 4, but these are infrequent, and comprehensive analyses are lacking. To reduce bias, we investigated many thermostable DNA polymerases and alternate reaction conditions for amplification of adapter-ligated fragments for Illumina sequencing. We expect this comparison to be relevant to other applications that involve simultaneous amplification of complex fragment mixtures.