Detection and quantification of rare mutations with massively parallel sequencing

The identification of mutations that are present in a small fraction of DNA templates is essential for progress in several areas of biomedical research. Although massively parallel sequencing instruments are in principle well suited to this task, the error rates in such instruments are generally too high to allow confident identification of rare variants. We here describe an approach that can substantially increase the sensitivity of massively parallel sequencing instruments for this purpose. The keys to this approach, called the Safe-Sequencing System (“Safe-SeqS”), are (i) assignment of a unique identifier (UID) to each template molecule, (ii) amplification of each uniquely tagged template molecule to create UID families, and (iii) redundant sequencing of the amplification products. PCR fragments with the same UID are considered mutant (“supermutants”) only if ≥95% of them contain the identical mutation. We illustrate the utility of this approach for determining the fidelity of a polymerase, the accuracy of oligonucleotides synthesized in vitro, and the prevalence of mutations in the nuclear and mitochondrial genomes of normal cells

FAST-SeqS: A Simple and Efficient Method for the Detection of Aneuploidy by Massively Parallel Sequencing

<div><p>Massively parallel sequencing of cell-free, maternal plasma DNA was recently demonstrated to be a safe and effective screening method for fetal chromosomal aneuploidies. Here, we report an improved sequencing method achieving significantly increased throughput and decreased cost by replacing laborious sequencing library preparation steps with PCR employing a single primer pair designed to amplify a discrete subset of repeated regions. Using this approach, samples containing as little as 4% trisomy 21 DNA could be readily distinguished from euploid samples.</p> </div

Demonstration of FAST-SeqS reproducibility among different samples, sequencing instruments, and sequencing depth.

<p>FAST-SeqS was performed on eight normal plasma DNA samples, their corresponding matched peripheral blood white blood cell (WBC) DNA, and on the splenic or WBC DNA of an additional eight unrelated individuals. The eight samples within each experiment constituted the reference group (see ‘Materials and Methods’ section) from which the plotted z-scores were calculated. No autosome in any sample had a z-score outside the range of −3.0 and 3.0 (dotted lines). Despite 3-fold less sequencing of the splenic or WBC samples, the z-scores (range: −2.2 to 2.1) were similar to those obtained from the plasma (range: −2.1 to 1.9) and matched WBC DNA samples (range: −2.2 to 1.9).</p

Accurate discrimination of euploid DNA samples from those containing trisomic DNA.

<p>(A) Comparison of z-scores from patients with trisomy 21 (n = 4), trisomy 18 (n = 2), and trisomy 13 (n = 1) with eight normal spleen or peripheral blood white blood cell (WBC) DNAs. The z-scores displayed represent the relevant chromosome for the comparison. The maximum z-score observed for any of the compared normal chromosomes was 1.9 (chr13). (B) Control WBC DNA was analyzed alone (n = 2) or when mixed with DNA from a patient with trisomy 21 at 5% (n = 2), 10% (n = 1), or 25% (n = 1) levels. A tight correlation existed between the expected and observed fractions of extra chromosome 21 (r = 0.997 by Pearson correlation test, n = 6). (C) Control WBC DNA was analyzed alone (z-score range: −0.8 to 1.3) or when mixed with DNA from a patient with trisomy 21 at 4% (z-score range: 4.5 to 7.2) or 8% (z-score range: 8.9 to 10.) levels. Each experiment in (C) was performed in quadruplicate.</p

Comparison of observed and predicted distributions of FAST-SeqS amplification products.

<p>(A) A density plot of the expected distribution of fragment lengths, with peaks at 124 and 142 bp. (B) A density plot of the actual tag counts obtained in eight normal plasma DNAs. The 124 bp fragments are preferentially amplified compared to the 142 bp fragments, likely due to an amplification bias towards smaller fragments. Inset: polyacrylamide gel of a representative FAST-SeqS sequencing library. Note: the amplification products contain an additional ∼120 bp of flanking sequence to facilitate sequencing (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041162#pone.0041162.s002" target="_blank">Table S2</a>). (C) The average representation of the most frequently observed L1 retrotransposon subfamilies in eight normal plasma samples. Roughly 97% of uniquely aligning tags arise from positions representing only seven L1 retrotransposon subfamilies. (D) A detailed examination of the average number of observed positions per chromosome from eight normal plasma DNAs compared with the number predicted by RepeatMasker for each of the seven L1 retrotransposon subfamilies noted in (C). Error bars in each panel depict the range.</p

Abstract NG01: Accumulation of somatic mutations in normal and cancerous tissues with age

Abstract Fundamental theories in carcinogenesis and aging evoke the accumulation of rare somatic mutations in normal tissues over time. However, absence of a simple and systematic method to characterize somatic mutations in normal tissues precludes the understanding of their functional consequences. We present Bottleneck Sequencing System (BotSeqS), a next-generation sequencing method that quantitates rare somatic point mutations simultaneously across the mitochondrial and nuclear genomes. BotSeqS combines molecular barcoding with a simple dilution step immediately before library amplification. Using BotSeqS, we determine the mutation frequencies and spectra in normal brain, kidney, and colon from a total of 34 individuals ranging from &amp;lt; 1 to 98 years old. We show an age and tissue-dependent accumulation of rare point mutations and demonstrate that the somatic mutational burden in normal tissues can vary by several orders of magnitude depending on biological and environmental factors. For example, individuals with defects in the mismatch repair machinery or exposure to environmental carcinogens (smoking, aristolochic acid) show significant increases in mutational frequencies and altered mutational spectra compared to controls. We further show major differences between the mutational patterns of the mitochondrial and nuclear genomes in normal tissues. Lastly, we find that the mutational spectra of normal tissues were different from each other but similar to cancers from the same tissue type, suggesting that the differential mutational spectra observed in cancers are tissue-specific rather than cancer-specific. This technology can provide insights into the number and nature of genetic alterations in normal tissues and can be used to address a variety of fundamental questions about the genomes of diseased tissues. Citation Format: Margaret L. Hoang, Isaac Kinde, Cristian Tomasetti, Thomas Rosenquist, Arthur P. Grollman, Kenneth W. Kinzler, Bert Vogelstein, Nickolas Papadopoulos. Accumulation of somatic mutations in normal and cancerous tissues with age. [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 NG01.</jats:p

The potential of circulating tumor DNA (ctDNA) to reshape the design of clinical trials testing adjuvant therapy in patients with early stage cancers

BACKGROUND: The conventional approach to testing the benefit of adjuvant therapies in patients (pts) with relatively favorable prognoses is to follow a large number of pts for long periods of time, hoping that mature outcome data will document an improved outcome compared to control pts. We reasoned that the design of such trials could be improved if pts with minimal residual disease could be identified a priori through the presence of ctDNA, and the effects of adjuvant therapy then assessed through serial ctDNA assays. METHODS: We carried out a prospective trial in 231 pts with Stage II colon cancer. Serial plasma samples were collected every 3 months starting 4-10 weeks after surgery. Somatic mutations in pts' tumors were identified via sequencing of 15 genes commonly mutated in colon cancer. We then designed personalized assays to quantify ctDNA in plasma samples. Adjuvant chemotherapy was administered at clinician discretion, blinded to ctDNA analysis. RESULTS: Somatic mutations were identified in 230 (99.6%) of tumors. Matching ctDNA was detected in the immediate post-operative period in 14 of 178 (8%) pts not treated with chemotherapy, 11 of whom had recurred (79%) at a median follow-up of 27 months. In contrast, recurrence occurred in only 16 (10%) of the 164 pts with negative ctDNA not treated with chemotherapy (HR 15.66, log-rank P<0.0001). ctDNA was detected in the immediate post-operative period in 6 of 52 pts who went on to receive chemotherapy. The ctDNA status turned from positive to negative during adjuvant treatment phase in all 6 pts (100%) but became positive again following completion of chemotherapy in 2 pts, both of whom have recurred. In patients with serial samples available, the median lead-time between ctDNA detection and radiologic-recurrence was 167 days. CONCLUSIONS: Detection of ctDNA in pts with resected stage II colon cancer provides direct evidence of residual disease. As well as defining pts at very high risk of later radiologic-recurrence, serial ctDNA analysis may provide an early readout of adjuvant treatment effect. Including ctDNA analyses would increase the efficiency of clinical trials testing the benefit of adjuvant treatment

Genome-wide quantification of rare somatic mutations in normal human tissues using massively parallel sequencing

Significance While we age, our body accumulates random somatic mutations. These mutations spontaneously arise from endogenous and exogenous sources, such as DNA replication errors or environmental insults like smoking or sunlight. Direct measurement of rare mutations could help us understand the role of somatic mutations in human aging, normal biology, and disease processes. Here, we develop the bottleneck sequencing system (BotSeqS) as a simple genome-wide sequencing-based method that accurately quantitates nuclear and mitochondrial mutational load in normal human tissues. We demonstrate that mutation prevalence and spectrum vary depending on age, tissue type, DNA repair capacity, and carcinogen exposure. Our results suggest a varied landscape of rare mutations within the human body that has yet to be explored.</jats:p