SimpliFi HS Mix is a high-fidelity, high-efficiency hot-start PCR master mix for NGS workflows requiring DNA library amplification prior to sequencing 1 . The fidelity of the SimpliFi HS Mix was determined using single-molecule sequencing and compared to commercially available high-fidelity master mixes. The results are demonstrating the exceptional fidelity of SimpliFi HS Mix, offering higher level of confidence for preserving DNA sequence accuracy during the preparation of DNA for next-generation sequencing.
Fidelity comparison across commercially available high-fidelity polymerases
Purified plasmid DNA was extended using SimpliFi, the complementary strands were synthesized using a standard high-fidelity polymerase, in both cases using primers containing a partial Illumina adapter, a random product barcode and a condition barcode. Primers complementary to the partial Illumina adapter are used to PCR amplify the complementary strands, forming the sequencing library. After next-generation sequencing, reads are grouped according to condition barcode and product barcode. Sequences are aligned to the correct sequence and errors are called. Errors are only kept if they are present in all copies, otherwise they are discarded as sequencing error. Exactly the same method was used to determine the error rate for Q5 (NEB), Kapa HiFi HotStart ReadyMix (Roche) and Phusion HS Flex (NEB), and the fidelity values were normalized to Taq polymerase fidelity.
Optimized for low GC bias PCR amplification can be susceptible to bias resulting from genomes that contain unusually high or low GC content, SimpliFi HS Mix has been developed to address this through careful selection of high-quality polymerase and optimization of the buffer, making it ideal for NGS library preparation.
GC bias with both high and low GC content
Libraries were prepared from 100 ng (amplified libraries) or 1 μg (PCR-free library) of genomic DNA from S. aureus (32% GC) and R. spheroides (69% GC), mixed equimolarly. End-repair and ligation steps were carried out using JetSeq™ ER and Ligation Kit (BIO-68026) and for all the samples. Library amplification consisted of 10 PCR cycles using SimpliFi HS Mix and Kapa HiFi HotStart ReadyMix (Roche), following the manufacturer’s recommendations. GC bias plots were generated, with %GC content of 100 bp windows on the X axis. Normalized coverage is indicated for the High- SimpliFi HS Mix ( blue line ) and Kapa HiFi ( green line ) and can be compared to a PCR-free reference library ( black line ), which has the horizontal bias distribution closer to 1 (representing unbiased coverage). The results illustrate SimpliFi HS Mix has a lower bias across the entire range of GC content, compared to the PCR- amplified library using Kapa HiFi HotStart ReadyMix (Roche) especially for GC percentages higher than 65%.
Supplier K | SimpliFi | PCR-free Reference
Applications: • Gene expression • Viral and bacterial detection • Robust PCR • High-specificity PCR
PRODUCT
CAT NO.
REACTIONS
BIO-25060 100 Reactions BIO-25061 500 Reactions
SimpliFi HS Mix (2x)
To review the product in detail or download our flyer, visit: https://www.bioline.com/simplifi-hs-mix-2x.html
• High-fidelity PCR • GC/AT-rich PCR
References 1. Lee D.F., et al. (2016) Mapping DNA polymerase errors by single-molecule sequencing. Nucleic Acids Res. 44(13): e118; PMID: 27185891
www.bioline.com
| 5 Quarterly newsletter
Powered by FlippingBook