Introduction: Minimally invasive liquid biopsies, which enable the characterization of circulating tumor DNA (ctDNA), are increasing in popularity and have the potential to improve patient management and outcomes.  The diversity in methods to isolate, capture and interpret ctDNA calls for improvement and standardization of sample extraction and library preparation methods, to enable unbiased interpretation of data. Here we present a method for the efficient capture of small (sub-nucleosomal) ctDNA fragments in an Illumina compatible format and for spike-in standards that will allow for improved detection in liquid biopsy assays.

Methods: A protocol for preparing small insert libraries (≥ 50 bp inserts) was developed by constructing libraries with double stranded oligonucleotides of 10 bp, 15 bp, 25 bp, 50 bp, and 90 bp and ensuring that adaptor dimers were eliminated from the final libraries.  We then constructed 1 ng cell free DNA libraries (BioChain custom cfDNA extraction) with this protocol to show that DNA fragments as small as 50 bp were captured and that the libraries contained no adaptor dimers, were complex and represented the human reference sequence.  Finally, we constructed small insert NGS libraries with Seraseq ctDNA Complete Mutation Mix AF5%, AF1% and AF0.1% (SeraCare Life Sciences) that contained fragmented human DNA with spiked-in amplicons that represent 25 unique multiplexed variants in 16 genes for each frequency.  All libraries were quantified by Qubit (ThermoFisher), analyzed on a Bioanalyzer (Agilent) and sequenced on an Illumina NextSeq instrument.

Results: Successful NGS libraries were constructed using double-stranded oligonucleotides of 15, 25, 50 and 90 bp and subsequent Bioanalyzer analysis showed no detectable adaptor dimer.  Libraries constructed with 1 ng of cfDNA (BioChain custom extraction) also showed no adaptor dimer with Bioanalyzer analysis and sequencing data showed complex libraries with uniform genome coverage.  Finally, we were able to detect variants down to 5% frequency using 1 ng of input DNA into the small insert library preparation protocol.

Conclusions:  We have developed a small insert library preparation protocol that allows for the construction of adaptor-dimer-free NGS libraries from DNA inserts as small as 50 bp.  We have demonstrated the utility of this protocol by constructing and analyzing cfDNA libraries and libraries prepared using Seraseq ctDNA Complete Mutation Mixes. We are developing a single-tube, ctDNA standard with different allelic frequencies that will control for sample extraction, NGS library preparation and sequence analysis.

Introduction: Commercially available RTs used in molecular biology are generally derived from Moloney murine leukaemia virus (MMLV) or avian myeloblastosis virus (AMV), which have optimal activity at 37-42 °C. These and most commercially available engineered RTs have limited activity at, and tolerance to, higher temperatures, and suffer limited stability at ambient temperature and on automated handling platforms. Higher reaction temperatures enable detection of difficult RNA templates by melting secondary structures. In this study, we compared RapiDxFire Thermostable Reverse Transcriptase thermostability, speed, and sensitivity to two commercial thermostable RTs. 

Methods: Reaction temperature and sensitivity were evaluated using a two-step real-time RT-qPCR process. In separate reactions, each enzyme was used to create cDNA from Zika virus or Human total RNA at RT reaction temperatures of 55, 60, and 65 °C for 10 minutes, and the cDNA was evaluated by real-time qPCR. Stability of reverse transcriptases was first determined in storage buffer by storing enzyme aliquots at ambient temperature and -20 °C for 7, 14, 42, 77, and 90 days. RapiDxFire synthesis of first-strand cDNA from MS2 RNA was measured via real-time qPCR using two MS2 primers. Stability was evaluated directly based on the increase in Cq value or indirectly by the decrease in cDNA synthesis. Thermotolerance of reverse transcriptases under reaction conditions was determined by pre-incubating reaction mixes containing each enzyme for 0, 15, 30, 45, and 60 minutes at 55, 60, and 65 °C.. RT reactions containing nucleotide and template/primer were then incubated for 15 minutes at 55 °C before quantifying the RNA/cDNA product by PicoGreen dye to measure polymerisation activity. 

Results: RapiDxFire Thermostable RT provided consistent results across reaction temperatures with superior sensitivity compared to the other thermostable reverse transcriptases, was stable for at least 3 months at room temperature, and was the only RT that remained stable under 1x reaction conditions for extended periods of time at 55, 60, and 65 °C. 

Conclusion: RapiDxFire Thermostable Reverse Transcriptase is a unique enzyme that provides superior thermostability when compared to other commercial reverse transcriptases, allowing for higher reaction temperatures, improved cDNA yield and specificity from difficult RNA targets. It also offers fast reaction times and flexible storage solutions, resulting in superior performance in applications where high temperature RNA synthesis is required.