Error Rate Of Next-generation Sequencing Research Articles

Lessons from discovery of true ADAR RNA editing sites in a human cell line

BackgroundConversion or editing of adenosine (A) into inosine (I) catalyzed by specialized cellular enzymes represents one of the most common post-transcriptional RNA modifications with emerging connection to disease. A-to-I conversions can happen at specific sites and lead to increase in proteome diversity and changes in RNA stability, splicing, and regulation. Such sites can be detected as adenine-to-guanine sequence changes by next-generation RNA sequencing which resulted in millions reported sites from multiple genome-wide surveys. Nonetheless, the lack of extensive independent validation in such endeavors, which is critical considering the relatively high error rate of next-generation sequencing, leads to lingering questions about the validity of the current compendiums of the editing sites and conclusions based on them.ResultsStrikingly, we found that the current analytical methods suffer from very high false positive rates and that a significant fraction of sites in the public databases cannot be validated. In this work, we present potential solutions to these problems and provide a comprehensive and extensively validated list of A-to-I editing sites in a human cancer cell line. Our findings demonstrate that most of true A-to-I editing sites in a human cancer cell line are located in the non-coding transcripts, the so-called RNA 'dark matter'. On the other hand, many ADAR editing events occurring in exons of human protein-coding mRNAs, including those that can recode the transcriptome, represent false positives and need to be interpreted with caution. Nonetheless, yet undiscovered authentic ADAR sites that increase the diversity of human proteome exist and warrant further identification.ConclusionsAccurate identification of human ADAR sites remains a challenging problem, particularly for the sites in exons of protein-coding mRNAs. As a result, genome-wide surveys of ADAR editome must still be accompanied by extensive Sanger validation efforts. However, given the vast number of unknown human ADAR sites, there is a need for further developments of the analytical techniques, potentially those that are based on deep learning solutions, in order to provide a quick and reliable identification of the editome in any sample.

The Clonal Hematopoietic Spectrum of Down Syndrome and ML-DS

Introduction: Children with Down syndrome (DS) have a 150-fold higher risk of developing myeloid leukemia (ML-DS) (Wechsler J, Nat Gen 2002). Ten percent (10%) of DS children are also predisposed to developing a preleukemic self-limiting condition, transient myeloproliferative disorder (TMD). While the majority of these children achieve complete remission, 20-30% of TMD patients will eventually develop ML-DS (Gamis AS, Blood 2011). Unique to ML-DS and TMD is the association with mutations in the X-linked hematopoietic transcription factor, GATA1 (Alford KA, Blood 2011), which are pathognomonic, but perhaps not sufficient, for ML-DS. Along with GATA1 mutations, ML-DS has a distinct mutational landscape implicating the cohesin complex, epigenetic regulators, signal transducers and the RAS pathway (Yoshida K, Nat Gen 2013). While these findings have informed our understanding of ML-DS etiology, ~30% of healthy DS neonates - without overt TMD or ML-DS - also harbor GATA1 mutations (Roberts I, Blood 2013), suggesting that ML-DS arises from clonal outgrowth of these progenitors that acquire specific additional mutations. Because the spectrum of physiologic clonal hematopoiesis is unknown in DS, we sought to characterize clonal mutations in healthy DS children as compared to a cohort of ML-DS cases using our highly sensitive and specific error-corrected sequencing (ECS) methodology. A precise understanding of their altered hematopoietic development may inform risk stratification, therapeutic selection and outcomes.Methods: The Druley lab has developed a custom error-corrected sequencing (ECS) panel targeting 80 genes frequently mutated in both pediatric and adult myeloid malignancies (Young AL, Nat Comm 2016) that is validated to identify clonal mutations as rare as 0.0001 variant allele fraction (VAF), 100X below the error-rate of next-generation sequencing (Wong TN, Nature 2015; Young AL, Leukemia 2015). Using ECS, we surveyed 102 DS children (47 ML-DS enrolled from a current Phase III ML-DS study; 55 healthy DS enrolled at St. Louis Children's Hospital).Results: We identified 294 total clonal variants in 72/102 children (60.0%, healthy DS; 83.0%, ML-DS cases) at 0.0002-0.82 VAF. On average, we found 1.8 clonal variants per healthy DS control and 4.1 clonal variants per ML-DS case. Consistent with Yoshida et al., we find a spectrum of recurrent mutations in ML-DS cases: GATA1 (53.2%), EZH2 (25.5%), RAD21 (14.9%), and STAG2 (10.6%) etc. Surprisingly, we also identified frequent mutations in FAT1 (14.9%) and SETD2 (10.6%), genes previously unassociated with ML-DS. Moreover, we report a different set of recurrent mutations in healthy DS children: FAT1 (12.7%), BCOR (12.7%), TET2 (10.9%), SETD2 (7.3%), and TRIM24 (5.5%), and TP53 (5.5%) etc.Discussion: Earlier ML-DS studies could only detect more common mutations >0.02 VAF due to the error-rate of standard next-generation sequencing (NGS). Using ECS, we present the first characterization of the unique clonal hematopoietic spectrum in children with DS and ML-DS. These results reveal that clonal hematopoiesis is common amongst healthy DS children without hematologic conditions, and that children with ML-DS have a higher clonal mutation burden than those without. Notably, we identified two novel genes recurrently mutated in both ML-DS cases and healthy DS controls: FAT1 and SETD2. We suspect that these mutations were not identified in other ML-DS studies as the median VAFs of our detected FAT1 and SETD2 mutations were 0.001 and 0.0008, respectively. Mutations in FAT1 and SETD2 have been shown to lead to dysregulated Wnt signaling in numerous cancers (Morris LG, Nat Gen 2013; Yuan H, J Clin Invest 2017). We and others have previously shown that Wnt signaling is crucial in specifying a primitive or definitive hematopoietic program (Sturgeon CM, Nat Biotech 2014; Creamer JP, Blood 2017). We speculate that these mutant FAT1 and SETD2 clones, like mutant GATA1 clones, may inhibit the definitive hematopoietic potential in DS children. Lastly, we note a reduced incidence of GATA1 mutations in our healthy DS cohort and attribute this to an older average age (8.3 years), as compared to Roberts et al. studying neonates. Other groups have speculated that somatic mutations in GATA1 (with a DS background) may only occur during a restricted developmental window (<4 years) as TMD clones cycle into quiescence afterwards (Zhe L, Nat Gen 2005; Hasle H, Leukemia 2007). DisclosuresNo relevant conflicts of interest to declare.

Abstract 3377: Extensive subclonal mutations in human colorectal cancers detected by duplex sequencing

Abstract The accumulation of somatic mutations is a defining hallmark of cancer. Human colorectal cancers (CRC) contain thousands of mutations that are detected at frequencies greater than 1%. Some of these are “driver mutations” that have been positively selected during tumor progression. In addition, subclonal mutations—those occurring only in a fraction of malignant cells—are randomly distributed and contribute to phenotypic and morphologic heterogeneity of cancer cells within a tumor. Cells containing specific subclonal mutations may be present prior to therapy and can be positively selected and account for the rapid emergence of therapeutic resistance. The extent of subclonal mutations in cancer, however, has been difficult to quantify, as the high error rate of next-generation sequencing precludes reliable detection of mutations present in fewer than 1-5% of cells. Here, we apply the highly accurate duplex sequencing methodology to quantify the extent of subclonal mutations in CRC. We analyzed somatic mutations in genes encoding replicative DNA polymerases (POLδ and POLε) as well as in genes reported to be frequently mutated in CRC. Duplex sequencing was carried out at depths varying from 500X-30,000X and an accuracy of 10-8. We find that CRCs with DNA repair deficits harbor an unexpectedly large complement of unique subclonal mutations; indicating that the mutational diversity of CRCs has been greatly underestimated. The frequency of subclonal mutations in normal colonic mucosa and CRC is greater than reported for clonal mutations. The burden of tumor-associated subclonal mutations does not correlate with age and the spectrum of single-nucleotide substitutions is different from that in nonmalignant cells, indicating that different mechanisms of mutation accumulation are operative in normal and CRC. Our data are consistent with a mutation rate of 1.1 x 10 per base pair, indicating that each malignant cell and its daughter encoded genomic DNA differs by more 3,000 nucleotides. The linearity of subclonal mutation accumulation as a function of sequencing depth, using DNA obtained from five different tumors, is in accord with a neutral model of tumor evolution. We calculate that the probability of mutations at any type is so high that it is likely that every possible somatic point mutation is present by the time a tumor is clinically detectable, and this could account for the high frequency by which tumors become resistant to therapeutic agents. Citation Format: Lawrence A. Loeb, Kaitlyn J. Loubet-Senear, Brendan F. Kohrn, Michael W. Schmitt, Mary P. Bronner, Robert A. Beckman. Extensive subclonal mutations in human colorectal cancers detected by duplex sequencing [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 3377.

Abstract A08: Extensive subclonal mutations in human colorectal cancers detected by Duplex Sequencing

Abstract The accumulation of somatic mutations is a defining hallmark of cancer. The genomes of solid tumors contain thousands of mutations that are present in most or all of the malignant cells in that tumor. In addition to these clonal mutations, subclonal mutations, those occurring only in a fraction of malignant cells, likely contribute to the phenotypic and morphologic heterogeneity of cancer cells within a tumor. The extent of subclonal mutations in cancer, however, has been difficult to quantify, as the high error rate of next-generation sequencing precludes reliable detection of mutations present in fewer than 5% of cells. Here, we apply the highly accurate Duplex Sequencing methodology to quantify the extent of subclonal mutations in colorectal cancer (CRC) and paired normal mucosa. By sequencing known CRC driver and non-driver genes, we find that colorectal cancers without known DNA repair deficits harbor an extensive complement of subclonal mutations, in addition to the large number of clonal mutations previously identified. We show that normal colonic mucosa also accumulates substantial numbers of subclonal mutations in an age-dependent manner. The frequency of tumor-associated subclonal mutations, however, does not correlate with age, and has a spectrum distinct from that seen in normal tissue, indicating different mechanisms of mutation accumulation are operative in normal and tumor tissue. The frequency of tumor cells harboring unique subclonal mutations is so high that it is likely that every possible neutral somatic point mutation is present by the time a tumor is clinically detectable. We propose that these subclonal mutations likely accumulate in a series of punctuated bursts, making it highly likely that resistant subclones will emerge during chemotherapy. Citation Format: Edward John Paul Fox, Michael W. Schmitt, Kate S. Reid-Bayliss, Robert A. Beckman, Lawrence A. Loeb. Extensive subclonal mutations in human colorectal cancers detected by Duplex Sequencing. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr A08.

Analysis of Base-Position Error Rate of Next-Generation Sequencing to Detect Tumor Mutations in Circulating DNA.

Detecting single-nucleotide variations and insertions/deletions in circulating tumor DNA is challenging because of their low allele frequency. The clinical use of circulating tumor DNA to characterize tumor genetic alterations requires new methods based on next-generation sequencing. We developed a method based on quantification of error rate of each base position [position error rate (PER)]. To identify mutations, a binomial test was used to compare the minor-allele frequency to the measured PER at each base position. This process was validated in control samples and in 373 plasma samples from patients with lung or pancreatic cancer. Minimal mutated allele frequencies were 0.003 for single-nucleotide variations and 0.001 for insertions/deletions. Independent testing performed by droplet digital PCR (n = 231 plasma samples) showed strong agreement with the base-PER method (κ = 0.90). Targeted next-generation sequencing analyzed with the base-PER method represents a robust and low cost method to detect circulating tumor DNA in patients with cancer.

Characterization of Viral Populations by Using Circular Sequencing.

With the enormous sizes viral populations reach, many variants are at too low a frequency to be detected by conventional next-generation sequencing (NGS) methods. Circular sequencing (CirSeq) is a method by which the error rate of next-generation sequencing is decreased so that even low-frequency viral variants can be accurately detected. The ability to visualize almost the entire genetic makeup of a viral swarm has implications for epidemiology, viral evolution, and vaccine design. Here we discuss experimental planning, analysis, and recent insights using CirSeq.

Abstract LB-338: Extensive subclonal mutations in human colorectal cancers detected by duplex sequencing

Abstract The accumulation of somatic mutations is a defining hallmark of cancer. The genomes of solid tumors contain thousands of mutations that are present in most or all of the malignant cells in that tumor. In addition to these clonal mutations, subclonal mutations - those occurring only in a fraction of malignant cells - likely contribute to the phenotypic and morphologic heterogeneity of cancer cells within a tumor. These sub-populations may be the source for the rapid emergence of therapeutic resistance. The extent of subclonal mutations in cancer, however, has been difficult to quantify, as the high error rate of next-generation sequencing precludes reliable detection of mutations present in fewer than 5% of cells. Here, we apply the highly accurate Duplex Sequencing methodology to quantify the extent of subclonal mutations in 15 colorectal cancers (CRC) and paired normal mucosa. We also quantify the clonal somatic mutation load by exome sequencing. By sequencing known CRC driver and non-driver genes (depth &amp;gt;5,000X and accuracy &amp;gt;10-7), we find that colorectal cancers without known DNA repair deficits harbor an extensive complement of subclonal mutations, suggesting that mutational diversity of CRCs has been greatly underestimated. We show that normal colonic mucosa also accumulates substantial numbers of subclonal mutations, predominantly CG&amp;gt;TA transitions at CpG dinucleotides, in an age-dependent manner. However, the burden of tumor-associated subclonal mutations does not correlate with age and has a distinct spectrum (relative increase in TA&amp;gt;CG transitions) indicating different mechanisms of mutation accumulation are operative in normal and tumor tissue. The frequency of tumor cells harboring unique subclonal mutations is so high that it is likely that every possible neutral somatic point mutation is present by the time a tumor is clinically detectable This could account for the high frequency by which tumors become resistant to chemotherapeutic agents. Citation Format: Edward J.P. Fox, Michael W. Schmitt, Kate S. Reid-Bayliss, Robert A. Beckman, Lawrence A. Loeb. Extensive subclonal mutations in human colorectal cancers detected by duplex sequencing. [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 LB-338.

Ultra-precise detection of mutations by droplet-based amplification of circularized DNA

BackgroundNGS (next generation sequencing) has been widely used in studies of biological processes, ranging from microbial evolution to cancer genomics. However, the error rate of NGS (0.1 % ~ 1 %) is still remaining a great challenge for comprehensively investigating the low frequency variations, and the current solution methods have suffered severe amplification bias or low efficiency.ResultsWe creatively developed Droplet-CirSeq for relatively efficient, low-bias and ultra-sensitive identification of variations by combining millions of picoliter uniform-sized droplets with Cir-seq. Droplet-CirSeq is entitled with an incredibly low error rate of 3 ~ 5 X 10-6. To systematically evaluate the performances of amplification uniformity and capability of mutation identification for Droplet-CirSeq, we took the mixtures of two E. coli strains as specific instances to simulate the circumstances of mutations with different frequencies. Compared with Cir-seq, the coefficient of variance of read depth for Droplet-CirSeq was 10 times less (p = 2.6 X 10-3), and the identified allele frequency presented more concentrated to the authentic frequency of mixtures (p = 4.8 X 10-3), illustrating a significant improvement of amplification bias and accuracy in allele frequency determination. Additionally, Droplet-CirSeq detected 2.5 times genuine SNPs (p < 0.001), achieved a 2.8 times lower false positive rate (p < 0.05) and a 1.5 times lower false negative rate (p < 0.001), in the case of a 3 pg DNA input. Intriguingly, the false positive sites predominantly represented in two types of base substitutions (G- > A, C- > T). Our findings indicated that 30 pg DNA input accommodated in 5 ~ 10 million droplets resulted in maximal detection of authentic mutations compared to 3 pg (p = 1.2 X 10-8) and 300 pg input (p = 2.2 X 10-3).ConclusionsWe developed a method namely Droplet-CirSeq to significantly improve the amplification bias, which presents obvious superiority over the currently prevalent methods in exploitation of ultra-low frequency mutations. Droplet-CirSeq would be promisingly used in the identification of low frequency mutations initiated from extremely low input DNA, such as DNA of uncultured microorganisms, captured DNA of target region, circulation DNA of plasma et al, and its creative conception of rolling circle amplification in droplets would also be used in other low input DNA amplification fields.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2480-1) contains supplementary material, which is available to authorized users.

Primer ID Validates Template Sampling Depth and Greatly Reduces the Error Rate of Next-Generation Sequencing of HIV-1 Genomic RNA Populations

Validating the sampling depth and reducing sequencing errors are critical for studies of viral populations using next-generation sequencing (NGS). We previously described the use of Primer ID to tag each viral RNA template with a block of degenerate nucleotides in the cDNA primer. We now show that low-abundance Primer IDs (offspring Primer IDs) are generated due to PCR/sequencing errors. These artifactual Primer IDs can be removed using a cutoff model for the number of reads required to make a template consensus sequence. We have modeled the fraction of sequences lost due to Primer ID resampling. For a typical sequencing run, less than 10% of the raw reads are lost to offspring Primer ID filtering and resampling. The remaining raw reads are used to correct for PCR resampling and sequencing errors. We also demonstrate that Primer ID reveals bias intrinsic to PCR, especially at low template input or utilization. cDNA synthesis and PCR convert ca. 20% of RNA templates into recoverable sequences, and 30-fold sequence coverage recovers most of these template sequences. We have directly measured the residual error rate to be around 1 in 10,000 nucleotides. We use this error rate and the Poisson distribution to define the cutoff to identify preexisting drug resistance mutations at low abundance in an HIV-infected subject. Collectively, these studies show that >90% of the raw sequence reads can be used to validate template sampling depth and to dramatically reduce the error rate in assessing a genetically diverse viral population using NGS. Although next-generation sequencing (NGS) has revolutionized sequencing strategies, it suffers from serious limitations in defining sequence heterogeneity in a genetically diverse population, such as HIV-1 due to PCR resampling and PCR/sequencing errors. The Primer ID approach reveals the true sampling depth and greatly reduces errors. Knowing the sampling depth allows the construction of a model of how to maximize the recovery of sequences from input templates and to reduce resampling of the Primer ID so that appropriate multiplexing can be included in the experimental design. With the defined sampling depth and measured error rate, we are able to assign cutoffs for the accurate detection of minority variants in viral populations. This approach allows the power of NGS to be realized without having to guess about sampling depth or to ignore the problem of PCR resampling, while also being able to correct most of the errors in the data set.

Primer ID Validates Template Sampling Depth and Greatly Reduces the Error Rate of Next-Generation Sequencing of HIV-1 Genomic RNA Populations

Primer ID Validates Template Sampling Depth and Greatly Reduces the Error Rate of Next-Generation Sequencing of HIV-1 Genomic RNA Populations