Effects of sequencing fidelity on cloning efficiency

A common research application of these dsDNA fragments is cloning into a bacterial vector for the purpose of expressing a protein. Mutated species in a mixed population of dsDNA fragments can have negative effects on the results of an experiment. Mutations can lead to codon changes, frameshifts, and even complete expression failure. While all types of mutations are possible, single base substitutions, or deletions, randomly distributed through the population of dsDNA fragments tend to be the most common DNA errors observed from commercially available fragments (Figure 1). These errors are often expressed as the ratio of incorrect to correct bases and may be introduced from the oligonucleotides used to assemble the fragments or by the assembly process itself. Likewise, additional PCR amplification of the fragment or the cloning process itself may introduce errors. 

To compensate for these DNA sequence errors, researchers will often pick and sequence multiple clones or colonies of bacteria to ensure that their desired sequence can be found. Since the errors tend to be random in nature, the higher the inherent error rate, the more colonies a researcher needs to select to increase the probability of finding the desired sequence. The random nature of the errors also means that the length of the insert is a factor, as longer insert sizes increase the probability of a random error being present. Therefore, as the length of insert increases, so too must the number of colonies researchers screen. Increased screening time and costs add to the total time and cost of an experiment. In this paper, we will demonstrate how the proprietary synthesis and sequence error correction processes of IDT result in lower error rates and why these processes lead to a reduction in screening effort to find a correct clone when compared to alternative DNA synthesis providers in the market.

To explore the benefit this correction has on cloning performance, IDT ordered eight fragments with lengths ranging from 300 bp to 1750 bp from other leading DNA fragment suppliers. Then, IDT manufactured the same sequences as gBlocks Gene Fragments for sequences under 1 kb, and as gBlocks HiFi Gene Fragments for sequences over 1 kb using our standard manufacturing pipeline. To highlight the effect of sequence composition on error rates and cloning efficiency, sequences were designed at each length range with both balanced GC (median GC% of 50.2%) and lower GC (median GC% of 40.2%).

All fragments were diluted to a fixed concentration using a TE pH 8.0 buffer. A length verification was performed using capillary electrophoresis on an Agilent Fragment Analyzer (FA). Following length verification, the sample was prepped for sequencing using a Nextera™ XT DNA Library Preparation Kit (Illumina™) and run on an Illumina™ MiSeq™ instrument using a 2 x 150 read length to determine error rate. Error rate was calculated by dividing the total number of errors by the total number of sequenced bases.

All sequences were then cloned into a pUC based vector using a seamless cloning method, transformed, and then grown overnight on agar plates. Twenty-four colonies of each sequence were selected and prepared using a Nextera™ XT DNA Library Preparation Kit and run on an Illumina™ MiSeq™ instrument to determine their sequence; poor-quality reads were discarded.

After assessing the lengths of the individual fragments, the next step was to assess the sequence composition. Double-stranded fragments typically contain a mixed population. If the mixed population includes a significant truncated product, it can be detected by CE or by running on a gel. Commonly, fragments contain randomly distributed point mutations with any given mutation appearing only on a few molecules. To assess these mutations, we sequenced each fragment using an Illumina™ MiSeq™ instrument (Figure 4). 

The effect of lower GC content can be observed in the sequences from all three suppliers. The sequences containing lower GC content contained higher error rates when compared to the sequences containing balanced GC. In both scenarios, IDT fragments displayed the lowest average error rate. The average error rate for IDT fragments was ~1 in 6300, a 73% lower error rate than obtained with fragments from Supplier B. 

This head-to-head comparison against two other major suppliers demonstrates how the IDT proprietary synthesis, assembly, and correction processes result in fragments with lower error rates, in fact, as low as 1:6300. These error rates brought about improved functionality in cloning applications with an improvement in correct clones as high as 100%. This increase could lead to researchers being able to reduce the number of colonies they need to screen by as much as 50%.

By minimizing the number of colonies needed to be screened, IDT gene fragments can reduce the rework steps researchers need to perform due to expression failure. Gene construction should be a seamless step in a research workflow and should not be aggravated by supplier issues such as unwanted mutations and truncations. Researchers using IDT gene fragments for cloning applications can therefore advance their studies rapidly in a budget-friendly manner to meet increasingly shorter project demands.

More than decreased error rates, IDT gene fragments have a short turnaround time and can be shipped in as few as 2 business days from order acceptance. So, as a reliable provider of high-quality dsDNA, IDT supplies gene fragments ideal for setting up cloning experiments for a variety of length ranges.