Biosciences Single-molecule Real-time Sequencing Research Articles

Discrete measurements of RNA polymerase and reverse transcriptase fidelity reveal evolutionary tuning.

Direct methods for determining the fidelity of DNA polymerases are robust, with relatively little sample manipulation before sequencing. In contrast, methods for measuring RNA polymerase and reverse transcriptase fidelities are complicated by additional preparation steps that introduce ambiguity and error. Here, we describe a sequencing method, termed Roll-Seq, for simultaneously determining the individual fidelities of RNA polymerases and reverse transcriptases (RT) using Pacific Biosciences single molecule real-time sequencing. By using reverse transcriptases with high rolling-circle activity, Roll-Seq generates long concatemeric cDNA from a circular RNA template. To discern the origin of a mutation, errors are recorded and determined to occur within a single concatemer (reverse transcriptase error) or all concatemers (RNA polymerase error) over the cDNA strand. We used Roll-Seq to measure the fidelities of T7 RNA polymerases, a Group II intron-encoded RT (Induro), and two LINE RTs (Fasciolopsis buski R2-RT and human LINE-1). Substitution rates for Induro and R2-RT are the same for cDNA and second-strand synthesis while LINE-1 has 2.5-fold lower fidelity when performing second-strand synthesis. Deletion and insertion rates increase for all RTs during second-strand synthesis. In addition, we find that a structured RNA template impacts fidelity for both RNA polymerase and RT. The accuracy and precision of Roll-Seq enable this method to be applied as a complementary analysis to structural and mechanistic characterization of RNA polymerases and reverse transcriptases or as a screening method for RNAP and RT fidelity.

Detection and Quantitation of DNA Damage on a Genome-wide Scale Using RADAR-seq.

The formation and persistence of DNA damage can impact biological processes such as DNA replication and transcription. To maintain genome stability and integrity, organisms rely on robust DNA damage repair pathways. Techniques to detect and locate DNA damage sites across a genome enable an understanding of the consequences of DNA damage as well as how damage is repaired, which can have key diagnostic and therapeutic implications. Importantly, advancements in technology have enabled the development of high-throughput sequencing-based DNA damage detection methods. These methods require DNA enrichment or amplification steps that limit the ability to quantitate the DNA damage sites. Further, each of these methods is typically tailored to detect only a specific type of damage. RAre DAmage and Repair (RADAR) sequencing is a DNA sequencing workflow that overcomes these limitations and enables detection and quantitation of DNA damage sites in any organism on a genome-wide scale. RADAR-seq works by replacing DNA damage sites with a patch of modified bases that can be directly detected by Pacific Biosciences Single-Molecule Real Time sequencing. Here, we present three protocols that enable detection of thymine dimers and ribonucleotides in bacterial and archaeal genomes. Basic Protocol 1 enables construction of a reference genome required for RADAR-seq analyses. Basic Protocol 2 describes how to locate, quantitate, and compare thymine dimer levels in Escherichia coli exposed to varying amounts of UV light. Basic Protocol 3 describes how to locate, quantitate, and compare ribonucleotide levels in wild-type and ΔRNaseH2 Thermococcus kodakarensis. Importantly, all three protocols provide in-depth steps for data analysis. Together they serve as proof-of-principle experiments that will allow users to adapt the protocols to locate and quantitate a wide variety of DNA damage sites in any organism. © 2022 New England Biolabs. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Constructing a reference genome utilizing SMRT sequencing Basic Protocol 2: Mapping and quantitating genomic thymine dimer formation in untreated versus UV-irradiated E. coli using RADAR-seq Basic Protocol 3: Mapping and quantitating genomic ribonucleotide incorporation in wildtype versus ΔRNaseH2 T. kodakarensis using RADAR-seq.

Prediction of SARS-CoV-2 Variant Lineages Using the S1-Encoding Region Sequence Obtained by PacBio Single-Molecule Real-Time Sequencing.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is the causal agent of the COVID-19 pandemic that emerged in late 2019. The outbreak of variants with mutations in the region encoding the spike protein S1 sub-unit that can make them more resistant to neutralizing or monoclonal antibodies is the main point of the current monitoring. This study examines the feasibility of predicting the variant lineage and monitoring the appearance of reported mutations by sequencing only the region encoding the S1 domain by Pacific Bioscience Single Molecule Real-Time sequencing (PacBio SMRT). Using the PacBio SMRT system, we successfully sequenced 186 of the 200 samples previously sequenced with the Illumina COVIDSeq (whole genome) system. PacBio SMRT detected mutations in the S1 domain that were missed by the COVIDseq system in 27/186 samples (14.5%), due to amplification failure. These missing positions included mutations that are decisive for lineage assignation, such as G142D (n = 11), N501Y (n = 6), or E484K (n = 2). The lineage of 172/186 (92.5%) samples was accurately determined by analyzing the region encoding the S1 domain with a pipeline that uses key positions in S1. Thus, the PacBio SMRT protocol is appropriate for determining virus lineages and detecting key mutations.

Evaluation of Bacterial Contamination in Goat Milk Powder Using PacBio Single Molecule Real-Time Sequencing and Droplet Digital PCR

Goat milk powder is a nutritious and easy-to-store product that is highly favored by consumers. However, the presence of contaminating bacteria and their metabolites may significantly affect the flavor, solubility, shelf life, and safety of the product. To comprehensively and accurately understand the sanitary conditions in the goat milk powder production process and potential threats from bacterial contamination, a combination of Pacific Biosciences single molecule real-time sequencing and droplet digital PCR was used to evaluate bacterial contamination in seven goat milk powder samples from three dairies. Ten phyla, 119 genera, and 249 bacterial species were identified. Bacillus, Paenibacillus, Lactococcus, and Cronobacter were the primary genera. Bacillus cereus, Lactococcus lactis, Alkaliphilus oremlandii, and Cronobacter sakazakii were the dominant species. With droplet digital PCR, 6.3 × 104 copies per g of Bacillus cereus and 1.0 × 104 copies per g of Cronobacter spp. were quantified, which may increase the risk of food spoilage and the probability of foodborne illness and should be monitored and controlled. This study offers a new approach for evaluating bacterial contamination in goat milk powder and supplies a reference for the assessment of food safety and control of potential risk, which will be of interest to the dairy industry.

Base modifications affecting RNA polymerase and reverse transcriptase fidelity.

Ribonucleic acid (RNA) is capable of hosting a variety of chemically diverse modifications, in both naturally-occurring post-transcriptional modifications and artificial chemical modifications used to expand the functionality of RNA. However, few studies have addressed how base modifications affect RNA polymerase and reverse transcriptase activity and fidelity. Here, we describe the fidelity of RNA synthesis and reverse transcription of modified ribonucleotides using an assay based on Pacific Biosciences Single Molecule Real-Time sequencing. Several modified bases, including methylated (m6A, m5C and m5U), hydroxymethylated (hm5U) and isomeric bases (pseudouridine), were examined. By comparing each modified base to the equivalent unmodified RNA base, we can determine how the modification affected cumulative RNA polymerase and reverse transcriptase fidelity. 5-hydroxymethyluridine and N6-methyladenosine both increased the combined error rate of T7 RNA polymerase and reverse transcriptases, while pseudouridine specifically increased the error rate of RNA synthesis by T7 RNA polymerase. In addition, we examined the frequency, mutational spectrum and sequence context of reverse transcription errors on DNA templates from an analysis of second strand DNA synthesis.

Preclinical and Clinical Resistance Mechanisms To The Investigational Selective FLT3 Inhibitor PLX3397 In FLT3-ITD+ Acute Myeloid Leukemia (AML)

BackgroundActivating mutations [primarily internal tandem duplication (ITD) events] in FLT3 are detected in 30% of acute myeloid leukemia (AML). The clinically active FLT3 tyrosine kinase inhibitor (TKI) AC220 (quizartinib) has achieved complete remissions in relapsed/refractory FLT3-ITD+ AML patients in a phase II study (Cortes, et al and Levis et al, ASH 2012, abstracts 48 and 673) but is vulnerable to resistance-conferring mutations in the FLT3 kinase domain (KD). The F691L “gatekeeper” substitution was the most commonly detected mutation in an in vitro mutagenesis screen for AC220 resistance (Smith et al, Nature 2012). This mutation, and substitutions at the activation loop residue D835, have been associated with acquired clinical resistance to AC220 (Smith et al, Nature 2012; Alber et al, Leukemia 2013). Mutations at gatekeeper residues such as F691 have repeatedly surfaced as mediators of clinical resistance to TKIs. Identifying TKIs that retain activity against these substitutions has consistently proven challenging. PLX3397 is a potent and selective inhibitor of FMS, KIT and FLT3-ITD with a half-life of 20 hours in humans, resulting in µM steady-state plasma concentrations at the recommended phase II dose for AML patients. PLX3397 retains activity against the AC220-resistant FLT3-ITD/F691L mutant, but not against several D835 mutants (Smith et al., ASH 2011, abstract 764). In this study, we conducted a mutagenesis screen of FLT3-ITD and FLT3-ITD/F691L to identify single and compound mutations that confer resistance to PLX3397 and may cause acquired resistance in patients. ResultsPLX3397 inhibited the proliferation of BaF3/ FLT3-ITD cells at a concentration well below that achieved in patients (IC50 0.14 µM) and retained activity against cells expressing the FLT3-ITD/F691L mutant (IC50 0.350 µM). Other AC220-resistant mutants (D835V/Y/F and Y842C/H) conferred substantial cross-resistance to PLX3397 (∼50 to 400-fold shift in IC50 of FLT3-ITD; ranging from 7.2 to >10 µM).An in vitro mutagenesis screen of FLT3-ITD identified several mutations conferring resistance to PLX3397, including novel substitutions in 3 residues which conferred ≥10X resistance relative to FLT3-ITD: D835E/G/N, D839A/G and R845G (IC50s 1.4 to 4.1 µM). Given the in vitro activity of PLX3397 against the AC220-resistant F691L mutant, it is anticipated that PLX3397 will be administered to patients who acquire resistance to AC220 or sorafenib due to this mutation; a mutagenesis screen of FLT3-ITD/F691L was therefore conducted. We identified multiple KD mutations in FLT3-ITD/F691L conferring ≥10X resistance to PLX3397 (compared to FLT3-ITD) including several mutations in the FLT3 activation loop: D835H/G/E/N, D839A/G/N, N841K, Y842S, R845G (IC50s 1.6 to >10 µM), and 2 mutations in residues located in the tyrosine kinase domain 1 (TK1) domain: N676S, a residue previously implicated in clinical resistance to the FLT3 inhibitor PKC412 (IC50 2.8 µM), and M664I, a residue not previously linked to FLT3 inhibitor resistance (IC50 2.0 µM). While all identified mutants conferred some degree of resistance to PLX3397 in the absence of an F691L mutation, most conferred a higher degree of resistance in the setting of F691L, suggesting a cooperation between the gatekeeper residue and residues in the activation loop and TK1 domain that impacts PLX3397 binding.Finally, we conducted a preliminary analysis of samples from AML patients who relapsed after an initial response to PLX3397. Using Pacific Biosciences Single Molecule Real-Time Sequencing, we identified evolution of polyclonal FLT3 KD mutations at the D835 residue at the time of relapse in 2 patients, including, in one patient, novel PLX3397-resistant D835E/H mutations identified in our mutagenesis screen. Analysis of additional patient samples for single and compound resistant mutations is ongoing and will be presented. ConclusionsPLX3397 harbors promise for the treatment of FLT3-ITD+ AML, particularly for patients who have developed resistance to FLT3 TKIs due to the gatekeeper F691L mutation. However, a mutagenesis screen reveals PLX3397 is vulnerable to mutations in the FLT3 activation loop and TK1 domain. Patients acquire secondary FLT3 KD mutations at the time of resistance to PLX3397, confirming the mechanism of action of this clinically active FLT3 inhibitor. A multi-site phase I/II study of PLX3397 in FLT3-ITD+ AML is ongoing. Disclosures:Smith:Plexxikon Inc: Research Funding. Off Label Use: Unapproved drugs for AML: AC220 and PLX3397. Le:Plexxikon Inc: Employment. Zhang:Plexxikon Inc: Employment. West:Plexxikon Inc: Employment. Shah:Ariad Pharmaceuticals: Consultancy, Research Funding; Plexxikon Inc: Research Funding; Ambit Biosciences: Research Funding.