High-throughput DNA sequencing errors are reduced by orders of magnitude using circle sequencing

Abstract

A major limitation of high-throughput DNA sequencing is the high rate of erroneous base calls produced. For instance, Illumina sequencing machines produce errors at a rate of ∼0.1-1 × 10 −2 per base sequenced. These technologies typically produce billions of base calls per experiment, translating to millions of errors. We have developed a unique library preparation strategy, "circle sequencing," which allows for robust downstream computational correction of these errors. In this strategy, DNA templates are circularized, copied multiple times in tandem with a rolling circle polymerase, and then sequenced on any high-throughput sequencing machine. Each read produced is computationally processed to obtain a consensus sequence of all linked copies of the original molecule. Physically linking the copies ensures that each copy is independently derived from the original molecule and allows for efficient formation of consensus sequences. The circlesequencing protocol precedes standard library preparations and is therefore suitable for a broad range of sequencing applications. We tested our method using the Illumina MiSeq platform and obtained errors in our processed sequencing reads at a rate as low as 7.6 × 10 −6 per base sequenced, dramatically improving the error rate of Illumina sequencing and putting error on par with low-throughput, but highly accurate, Sanger sequencing. Circle sequencing also had substantially higher efficiency and lower cost than existing barcode-based schemes for correcting sequencing errors.next-generation sequencing | barcoding | rare variants

Cell-free DNA (cfDNA): Clinical Significance and Utility in Cancer Shaped By Emerging Technologies

“…The combination of in silico filtering and barcoding approaches allowed development of the workflow that had 15 times lower error rate than original CAPP-Seq approach. An interesting alternative to barcoding techniques capitalized on the increased read length possible with newer Illumina chemistry and suggested the use of rolling circle amplification to ensure redundant sequencing of individual DNA fragments (90). Briefly, DNA is fragmented to approximately 130 bp, denatured, circularized, and amplified using Phi29 polymerase.…”

“…Clearly, linearized fragments are excluded from subsequent rolling circle amplification and sequencing. This allowed the authors to achieve approximately 100-fold reduction in sequencing noise thereby improving specificity, but presumably at a cost to overall sensitivity due to the loss of some molecules prior to sequencing (90).…”

Technicalities and Glitches of Terminal Restriction Fragment Length Polymorphism (T-RFLP)

“…In addition, NGS is an emerging alternative of T-RFLP for community profiling and provide more indepth information but currently facing some critics. For example, rate of sequencing error in next generation sequencing is high than Sanger sequencing [51]. In addition, due to short read length it gives overestimate about microbial diversity and cannot use for species level resolution.…”

“…Kalia et al [52], Porwal et al [53] and Bhushan et al [54] used extensive database of 16S rRNA gene sequences and developed species specific framework, signature nucleotide and restriction digestion pattern to increase the taxonomic resolution in genus Clostridium, Bacillus, and Pseudomonas respectively. Different approaches used by Kalia et al [51] and Porwal et al [52] can be used to get more taxonomic resolution by designing the species specific framework for community analysis or for the study of effect of environmental perturbation on a particular species by T-RFLP or NGS. Although next generation sequencing is more attractive but at the same time it is cumbersome and costly as compared to T-RFLP.…”

The Impact of Collisions on the Ability to Detect Rare Mutant Alleles Using Barcode-Type Next-Generation Sequencing Techniques

“…5 This has led to increased interest recently in refining NGS techniques to reduce error rates and noise to allow for detection of verylow-frequency mutations. [6][7][8] Several of these refinement methods rely on barcoding-type techniques in which sample molecules are tagged with unique identifiers (UIDs), an exogenous random sequences of nucleotides, prior to amplification. The tagged amplicons are then sequenced and post hoc grouped by UID into families to create a consensus sequence representing the original sample molecule, filtering out errors introduced in the amplification and sequencing process.…”