LFMD: a new likelihood-based method to detect low-frequency mutations without molecular tags

As next generation sequencing (NGS) and liquid biopsy become more prevalent in clinical and research area, especially cancer diagnosis, targeted therapy guidance and disease surveillance, there is an increasing need for better methods to reduce cost and to improve sensitivity and specificity. Since the error rate of NGS is around 1%, it is difficult to identify mutations with frequency lower than 1% accurately and efficiently because of low Signal-to- Noise Ratio (SNR). Here we propose a likelihood-based approach, low-frequency mutation detector (LFMD), combining the advantages of duplex sequencing (DS) and bottleneck sequencing system (BotSeqS) to maximize utilization of duplicate sequenced reads. Compared with DS, the new method achieves higher sensitivity (improved ~16%), higher specificity and lower cost (reduced ~70%) without involving additional experimental steps, customized adapters and molecular tags. In addition, this method can also be used to improve sensitivity and specificity of other variant calling algorithms by replacing a step in traditional NGS analysis: removing polymerase chain reaction (PCR) duplication. Thus, LFMD can be a promising method used in genomic research and clinical fields.

[1]  Marcus Lewis,et al.  Reducing amplification artifacts in high multiplex amplicon sequencing by using molecular barcodes , 2015, BMC Genomics.

[2]  YONG CHEN,et al.  A conditional composite likelihood ratio test with boundary constraints , 2018 .

[3]  Xinjian Wang,et al.  Age-Related Accumulation of Somatic Mitochondrial DNA Mutations in Adult-Derived Human iPSCs. , 2016, Cell stem cell.

[4]  Anton Nekrutenko,et al.  Streamlined analysis of duplex sequencing data with Du Novo , 2016, Genome Biology.

[5]  Shicai Wang,et al.  COSMIC: the Catalogue Of Somatic Mutations In Cancer , 2018, Nucleic Acids Res..

[6]  R. Dimond,et al.  Social and ethical issues in mitochondrial donation. , 2015, British medical bulletin.

[7]  Brendan F. Kohrn,et al.  Detecting ultralow-frequency mutations by Duplex Sequencing , 2014, Nature Protocols.

[8]  Jesse J. Salk,et al.  Detection of ultra-rare mutations by next-generation sequencing , 2012, Proceedings of the National Academy of Sciences.

[9]  Gonçalo R. Abecasis,et al.  Assessing Mitochondrial DNA Variation and Copy Number in Lymphocytes of ~2,000 Sardinians Using Tailored Sequencing Analysis Tools , 2015, PLoS genetics.

[10]  G. Nappi,et al.  The role of glutamate in the pathophysiology of Parkinson's disease. , 1996, Functional neurology.

[11]  Lawrence D True,et al.  Sequencing small genomic targets with high efficiency and extreme accuracy , 2015, Nature Methods.

[12]  Arthur P. Grollman,et al.  Genome-wide quantification of rare somatic mutations in normal human tissues using massively parallel sequencing , 2016, Proceedings of the National Academy of Sciences.

[13]  D. Wallace,et al.  Mitochondrial DNA genetics and the heteroplasmy conundrum in evolution and disease. , 2013, Cold Spring Harbor perspectives in biology.

[14]  Dawei Li,et al.  The diploid genome sequence of an Asian individual , 2008, Nature.

[15]  Umberto De Marchi,et al.  MitoRS, a method for high throughput, sensitive, and accurate detection of mitochondrial DNA heteroplasmy , 2017, BMC Genomics.

[16]  M. Drton.  Likelihood ratio tests and singularities , 2007, math/0703360.

[17]  A. Heger,et al.  UMI-tools: modeling sequencing errors in Unique Molecular Identifiers to improve quantification accuracy , 2016, bioRxiv.

[18]  Cassandra B. Jabara,et al.  Accurate sampling and deep sequencing of the HIV-1 protease gene using a Primer ID , 2011, Proceedings of the National Academy of Sciences.

[19]  J. Troge,et al.  Tumour evolution inferred by single-cell sequencing , 2011, Nature.

[20]  Jeffrey A. Hussmann,et al.  High-throughput DNA sequencing errors are reduced by orders of magnitude using circle sequencing , 2013, Proceedings of the National Academy of Sciences.

[21]  M. Tarnopolsky,et al.  Digital PCR methods improve detection sensitivity and measurement precision of low abundance mtDNA deletions , 2016, Scientific Reports.

[22]  Hairong Duan,et al.  Benefits and Challenges with Applying Unique Molecular Identifiers in Next Generation Sequencing to Detect Low Frequency Mutations , 2016, PloS one.

[23]  A. von Haeseler,et al.  A coalescent approach to the polymerase chain reaction. , 1997, Nucleic acids research.

[24]  Jiang Li,et al.  MitoSeek: extracting mitochondria information and performing high-throughput mitochondria sequencing analysis , 2013, Bioinform..

[25]  James Hicks,et al.  Single cell sequencing approaches for complex biological systems. , 2014, Current opinion in genetics & development.

[26]  Scott R. Kennedy,et al.  Mitochondrial DNA mutations increase in early stage Alzheimer disease and are inconsistent with oxidative damage , 2016, Annals of neurology.