Measuring sequencer size bias using REcount: a novel method for highly accurate Illumina sequencing-based quantification

Abstract

Quantification of DNA sequence tags from engineered constructs such as plasmids, transposons, or other transgenes underlies many functional genomics measurements. Typically, such measurements rely on PCR followed by next-generation sequencing. However, PCR amplification can introduce significant quantitative error. We describe REcount, a novel PCR-free direct counting method. Comparing measurements of defined plasmid pools to droplet digital PCR data demonstrates that REcount is highly accurate and reproducible. We use REcount to provide new insights into clustering biases due to molecule length across different Illumina sequencers and illustrate the impacts on interpretation of next-generation sequencing data and the economics of data generation. Electronic supplementary material The online version of this article (10.1186/s13059-019-1691-6) contains supplementary material, which is available to authorized users.

A rapid, cost-effective tailed amplicon method for sequencing SARS-CoV-2

“…Despite observing negligible amounts of primer dimer products on the bioanalyzer trace, samples with N1 and N2 Ct values greater than 30 had as much as 50% primer dimer in the resulting sequencing reads. We have previously reported a substantial size bias on the MiSeq, which may help explain the preferential clustering and out-sized proportion of primer dimer reads present in the sequencing data for some samples [16]. While this issue can be overcome by increased sequencing depth, future optimizations aimed at reducing primer dimer contamination such as more stringent size selection or sequencing on an instrument with less size bias, such as the NovaSeq [16] could reduce this effect.…”

“…We have previously reported a substantial size bias on the MiSeq, which may help explain the preferential clustering and out-sized proportion of primer dimer reads present in the sequencing data for some samples [16]. While this issue can be overcome by increased sequencing depth, future optimizations aimed at reducing primer dimer contamination such as more stringent size selection or sequencing on an instrument with less size bias, such as the NovaSeq [16] could reduce this effect. Introduction of a bead clean-up step between the first and second PCRs can also help reduce the proportion of adapter dimers when using the tailed amplicon v2 protocol (Amy Kistler, personal communication).…”

A Rapid, Cost-Effective Tailed Amplicon Method for Sequencing SARS-CoV-2

“…Despite observing negligible amounts of primer dimer products on the bioanalyzer trace, samples with N1 and N2 Ct values greater than 30 had as much as 50% primer dimer in the resulting sequencing reads. We have previously reported a substantial size bias on the MiSeq, which may help explain the preferential clustering and out-sized proportion of primer dimer reads present in the sequencing data for some samples (15). While this issue can be overcome by increased sequencing depth, future optimizations aimed at reducing primer dimer contamination such as more stringent size selection or sequencing on an instrument with less size bias, such as the NovaSeq (15) could reduce this effect.…”

Deconvolution of nucleic-acid length distributions: a gel electrophoresis analysis tool and applications

“…Further evaluation using the plug-in examined differences in size distributions obtained both from analysis of the lane profiles and from MiSeq post-sequencing fragment-size distributions for the completed library (see ‘Sequencing protocol and bioinformatic analysis’ section). Although input- and output-size profiles for the MiSeq will show some distortion due to the known preference for smaller fragments during bridge amplification, measurement of input material with the plug-in allows prediction of conditions most likely to alter that pattern of eventual fragment densities (11,17–20). Figure 3 quantitatively shows the effect of increasing numbers of amplification cycles on the fragment-size distributions obtained from MiSeq output.…”