Laurence Ettwiller

DNA damage is a pervasive cause of sequencing errors, directly confounding variant identification

When is a mutation a true genetic variant? Large-scale sequencing studies have set out to determine the low-frequency pathogenic genetic variants in individuals and populations. However, Chen et al. demonstrate that many so-called low-frequency genetic variants in large public databases may be due to DNA damage. They scored libraries sequenced with and without a DNA damage–repairing enzymatic mix to assess the proportion of true rare variants. It remains to be seen how best to repair DNA before sequencing to provide more accurate assessments of mutation. Science, this issue p. 752 Damaged DNA may artificially increase the number of rare variants identified in large-scale sequencing projects. Mutations in somatic cells generate a heterogeneous genomic population and may result in serious medical conditions. Although cancer is typically associated with somatic variations, advances in DNA sequencing indicate that cell-specific variants affect a number of phenotypes and pathologies. Here, we show that mutagenic damage accounts for the majority of the erroneous identification of variants with low to moderate (1 to 5%) frequency. More important, we found signatures of damage in most sequencing data sets in widely used resources, including the 1000 Genomes Project and The Cancer Genome Atlas, establishing damage as a pervasive cause of sequencing errors. The extent of this damage directly confounds the determination of somatic variants in these data sets.

Global repositioning of transcription start sites in a plant-fermenting bacterium

Bacteria respond to their environment by regulating mRNA synthesis, often by altering the genomic sites at which RNA polymerase initiates transcription. Here, we investigate genome-wide changes in transcription start site (TSS) usage by Clostridium phytofermentans, a model bacterium for fermentation of lignocellulosic biomass. We quantify expression of nearly 10,000 TSS at single base resolution by Capp-Switch sequencing, which combines capture of synthetically capped 5′ mRNA fragments with template-switching reverse transcription. We find the locations and expression levels of TSS for hundreds of genes change during metabolism of different plant substrates. We show that TSS reveals riboswitches, non-coding RNA and novel transcription units. We identify sequence motifs associated with carbon source-specific TSS and use them for regulon discovery, implicating a LacI/GalR protein in control of pectin metabolism. We discuss how the high resolution and specificity of Capp-Switch enables study of condition-specific changes in transcription initiation in bacteria.

Genome Filtering for New DNA Biomarkers of Loa loa Infection Suitable for Loop-Mediated Isothermal Amplification.

Loa loa infections have emerged as a serious public health problem in patients co-infected with Onchocerca volvulus or Wuchereria bancrofti because of severe adverse neurological reactions after treatment with ivermectin. Accurate diagnostic tests are needed for careful mapping in regions where mass drug administration is underway. Loop-mediated isothermal amplification (LAMP) has become a widely adopted screening method because of its operational simplicity, rapidity and versatility of visual detection readout options. Here, we present a multi-step bioinformatic pipeline to generate diagnostic candidates suitable for LAMP and experimentally validate this approach using one of the identified candidates to develop a species-specific LAMP assay for L. loa. The pipeline identified ~140 new L. loa specific DNA repeat families as putative biomarkers of infection. The consensus sequence of one family, repeat family 4 (RF4), was compiled from ~ 350 sequences dispersed throughout the L. loa genome and maps to a L. loa-specific region of the long terminal repeats found at the boundaries of Bel/Pao retrotransposons. PCR and LAMP primer sets targeting RF4 specifically amplified L. loa but not W. bancrofti, O. volvulus, Brugia malayi, human or mosquito DNA. RF4 LAMP detects the DNA equivalent of one microfilaria (100 pg) in 25–30 minutes and as little as 0.060 pg of L. loa DNA (~1/1600th of a microfilaria) purified from spiked blood samples in approximately 50 minutes. In summary, we have successfully employed a bioinformatic approach to mine the L. loa genome for species-specific repeat families that can serve as new DNA biomarkers for LAMP. The RF4 LAMP assay shows promise as a field tool for the implementation and management of mass drug administration programs and warrants further testing on clinical samples as the next stage in development towards this goal.

Response to Comment on “DNA damage is a pervasive cause of sequencing errors, directly confounding variant identification”

Following the Comment of Stewart et al., we repeated our analysis on sequencing runs from The Cancer Genome Atlas (TCGA) using their suggested parameters. We found signs of oxidative damage in all sequence contexts and irrespective of the sequencing date, reaffirming that DNA damage affects mutation-calling pipelines in their ability to accurately identify somatic variations.

Abstract 1425: A multi-enzyme DNA repair mix improves library quality and sequencing accuracy in FFPE tumor samples

Next-generation sequencing (NGS) methods are used extensively to profile mutations present in diseased human tissues. These genomic approaches hold great promise for personalized medicine but sequencing accuracy is essential for proper patient diagnosis and determining a treatment plan. A common source of DNA for genomic profiling is formalin-fixed, paraffin-embedded (FFPE) tissue samples obtained from patient biopsy. FFPE DNA poses important challenges for preparing NGS libraries including low input amounts and poor DNA quality, resulting from extensive fixation- and storage-induced DNA damage. Additionally, these damage-induced sequencing artifacts raise the background level of mutations, making it difficult to discern true, low frequency, disease-causing variants from noise. We previously showed that a major fraction of somatic mutations described in publicly available datasets are due to such sequencing artifacts (Chen et al., Science 2017). Furthermore, we showed that enzymatic repair of DNA before library preparation improves the library quality and reduces background noise. We developed a second-generation DNA repair enzyme mix (V2) that efficiently repairs the most prevalent damage types found in FFPE DNA and further improves the quality and yield of NGS libraries. Additionally, we tested the efficacy of the V2 repair mix in improving sequencing accuracy for FFPE DNA samples obtained from different cancer tissues. We performed target enrichment on a panel of 151 cancer genes, deep sequenced, and performed variant analysis. For a subset of variants, we further validated our results using a droplet digital PCR (ddPCR) assay. Both methods showed that the V2 repair mix did not alter the overall frequency of variants identified, thus it did not introduce bias, but significantly improved the sequencing accuracy by reducing the number of false variant calls. Therefore, enzymatic repair is a critical first step in preparing FFPE DNA sequencing libraries, allowing more sensitive and robust detection of low frequency, disease variants. Citation Format: Pingfang Liu, Margaret Heider, Chen Song, Lixin Chen, Laurence Ettwiller, Lauren Higgins, Eileen Dimalanta, Theodore Davis, Thomas Evans. A multi-enzyme DNA repair mix improves library quality and sequencing accuracy in FFPE tumor samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1425.

Abstract 5360: DNA repair increases sequencing accuracy without altering actual mutation frequency in clinical samples

Targeted cancer therapy based on genomic alterations can be remarkably effective. Currently, cancer genome profiling using next generation sequencing (NGS) is routinely applied in cancer care to guide personalized treatment. The accuracy of this profiling directly impacts therapeutic choices and the outcomes of patient care. We previously showed that false positive variants are abundant and can account for a major fraction of identified somatic variations in publicly available datasets (doi: http://dx.doi.org/10.1101/070334). These false positive variants show signs of mutagenic DNA damage. We further demonstrated that enzymatic DNA repair increases sequencing quality by lowering damage-induced background noise. Therefore, enzymatic DNA repair has the potential to improve sequencing accuracy, avoiding incorrect somatic variant calls and consequently reducing incorrect diagnostic conclusions. In this study, we investigated whether enzymatic DNA repair introduces any bias to NGS libraries using analysis by droplet digital PCR (ddPCR) and deep sequencing. DNA Reference Standards containing multiple common cancer mutations (Horizon Discovery, Inc.) were spiked into formalin-fixed paraffin-embedded (FFPE) DNA isolated from tumor samples from different tissue types at defined frequencies (0.5-10% quantified by ddPCR). Genotyping of the FFPE DNA ensured that they were free of any of the spiked-in mutations. After DNA repair and library preparation, mutation frequencies were quantified by ddPCR, and compared to the mutation levels in input DNA and control libraries without repair. Deep sequencing of 151 cancer genes including these spike-ins showed no difference in mutation frequency for the spiked-in mutations between the control and repair groups. However, the number of false positive variant calls was reduced in the repair group. Our data demonstrates that DNA repair significantly increases sequencing accuracy without altering the frequency of actual mutations in tumor samples. Citation Format: Pingfang Liu, Lixin Chen, Laurence Ettwiller, Eileen Dimalanta, Theodore B. Davis, Thomas C. Evans. DNA repair increases sequencing accuracy without altering actual mutation frequency in clinical samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5360. doi:10.1158/1538-7445.AM2017-5360

Abstract 3628: Improving sequencing quality of libraries prepared from FFPE DNA

Targeted cancer therapy based on genomic alterations can be remarkably effective, and has made significant strides with the recent advances in next-generation sequencing (NGS) technology. Although samples of blood and other bodily fluids are being actively explored for early disease diagnosis and treatment monitoring, DNA isolated from FFPE samples is currently the main source for NGS-based cancer profiling in clinical settings. Unfortunately, sequencing DNA from FFPE samples is challenging due to limited quantities and poor quality, a result of DNA damage incurred during fixation and storage. Artifacts associated with FFPE DNA have limited the mutation detection sensitivity to ≥ 5% mutant allele frequency (Frampton et al, Nature Biotechnology 2013), which would unfortunately leave many low-abundance genetic variants of clinical significance undetected. For example, clinical resistance-causing KIT and EGFR mutations can be present in tumors at levels In this study, we investigated the effects of DNA repair and different sample handling workflows on sequencing quality of libraries prepared from FFPE samples. Careful analysis of sequencing data showed that base calling qualities for all 4 bases are improved, and aberrant G:C to A:T mutations were significantly reduced upon DNA repair. Because the large majority of mutations encountered in human tumors are G:C to A:T mutations (Greenman, C. Nature 2007), we expect that lowering the damage induced background noise of FFPE DNA would allow more reliable detection of clinically important, actionable mutations at lower abundance. In addition, we observed specific sequencing artifacts associated with the method of handling FFPE samples and have since identified effective measurements to avoid such artifacts. We expect that these improvements in sequencing quality of FFPE samples would ultimately enable more sensitive and robust detection of many low level genetic variations in clinically and biologically relevant cancer genes. Citation Format: pingfang liu, Lixin Chen, Laurence Ettwiller, Christine Sumner, Fiona J. Stewart, Eileen T. Dimalanta, Theodore B. Davis, Evans C. Thomas. Improving sequencing quality of libraries prepared from FFPE DNA. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3628.