DOI: 10.5061/dryad.jj4g11s
title: A Streamlined and High-Throughput Error-Corrected Next-Generation Sequencing Method for Low Variant Allele Frequency Quantitation

description: Quantifying mutant or variable allele frequencies (VAFs) of ≤10−3 using
 next-generation sequencing (NGS) has utility in both clinical and
 nonclinical settings. Two common approaches for quantifying VAFs using NGS
 are tagged single-strand sequencing and duplex sequencing. While duplex
 sequencing is reported to have sensitivity up to 10−8 VAF, it is not a
 quick, easy, or inexpensive method. We report a method for quantifying
 VAFs that are ≥10−4 that is as easy and quick for processing samples as
 standard sequencing kits, yet less expensive than the kits. The method was
 developed using PCR fragment-based VAFs of Kras codon 12 in log10
 increments from 10−5 to 10−1, then applied and tested on native genomic
 DNA. For both sources of DNA, there is a proportional increase in the
 observed VAF to input VAF from 10−4 to 100% mutant samples. Variability of
 quantitation was evaluated within experimental replicates and shown to be
 consistent across sample preparations. The error at each successive base
 read was evaluated to determine if there is a limit of read length for
 quantitation of ≥10−4, and it was determined that read lengths up to 70
 bases are reliable for quantitation. The method described here is
 adaptable to various oncogene or tumor suppressor gene targets, with the
 potential to implement multiplexing at the initial tagging step. While
 easy to perform manually, it is also suited for robotic handling and batch
 processing of samples, facilitating detection and quantitation of genetic
 carcinogenic biomarkers before tumor formation or in normal-appearing
 tissue.
description: READMEA readme file that
 explains the information in the data files, some portions of the
 computational workflow, and the directory structure for replication of the
 computational workflow.illustration_tagging_computingAn scalar vector illustration file of the workflow made using Inkscape 0.92.schema_of_filesA text file describing the directory structure of the workflow.samples_replicate_numbersA two-column csv file indicating the sample name and replicate number for the appropriate experiment type (PCR-based or gDNA-based template).samples_metadataA csv file containing information on sample name, sample type, input variant allele fraction, observed variant allele fraction, and other information on various data file locations within the directory structure. All headers are explained in the README file.dir_structure.tarA tar 1.26 compressed file of the directory structure of the computational workflow for ease of replication. The subdirectories are empty of files. The use of this file is explained in the README file.dir_structure.gz.targDNA_stds_error_posA csv file documenting the observed error at targeted position (position 10 of reference) for each gDNA sample investigated.KRAS_WT_144144_nuc_countsA csv file containing the nucleotide counts for each position of gDNA WT sample, replicate 1.KRAS_WT_145145_nuc_countsA csv file containing the nucleotide counts for each position of gDNA WT sample, replicate 2.KRAS_10-5_144145_nuc_countsA csv file containing the nucleotide counts for each position of gDNA 10^-5 sample, replicate 1.KRAS_10-5_145146_nuc_countsA csv file containing the nucleotide counts for each position of gDNA 10^-5 sample, replicate 2.KRAS_10-4_144146_nuc_countsA csv file containing the nucleotide counts for each position of gDNA 10^-4 sample, replicate 1.KRAS_10-4_145147_nuc_countsA csv file containing the nucleotide counts for each position of gDNA 10^-4 sample, replicate 2.KRAS_10-3_144147_nuc_countsA csv file containing the nucleotide counts for each position of gDNA 10^-3 sample, replicate 1.KRAS_10-3_145148_nuc_countsA csv file containing the nucleotide counts for each position of gDNA 10^-3 sample, replicate 2.KRAS_10-2_144148_nuc_countsA csv file containing the nucleotide counts for each position of gDNA 10^-2 sample, replicate 1.KRAS_10-2_145149_nuc_countsA csv file containing the nucleotide counts for each position of gDNA 10^-2 sample, replicate 2.KRAS_10-1_144149_nuc_countsA csv file containing the nucleotide counts for each position of gDNA 10^-1 sample, replicate 1.KRAS_10-1_145150_nuc_countsA csv file containing the nucleotide counts for each position of gDNA 10^-1 sample, replicate 2.KRAS_MUT_144150_nuc_countsA csv file containing the nucleotide counts for each position of gDNA mutant only sample, replicate 1.KRAS_MUT_145151_nuc_countsA csv file containing the nucleotide counts for each position of gDNA mutant only sample, replicate 2.gDNA_all_group_size_histoA csv file containing data describing the number of groups with specified number of reads in the group for the gDNA samples. The first column indicates the number of reads, the remaining column headers are the sample names, and the list of numbers under each name is the number of groups of specified read depth.gDNA_all_group_read_lens_histoA csv file of the read lengths for each gDNA sample, with length as the row name and the number of reads with specified length for each sample across the columns. Column headers are the names of the samples.KRAS_WT_144144_read_qualsA csv documenting the median qualities of each position for each tag group in gDNA WT sample, replicate1. The header information is in the README.txt file.KRAS_WT_145145_read_qualsA csv documenting the median qualities of each position for each tag group in gDNA WT sample, replicate2. The header information is in the README.txt file.KRAS_10-5_144145_read_qualsA csv documenting the median qualities of each position for each tag group in gDNA 10^-5 sample, replicate1. The header information is in the README.txt file.KRAS_10-5_145146_read_qualsA csv documenting the median qualities of each position for each tag group in gDNA 10^-5 sample, replicate2. The header information is in the README.txt file.KRAS_10-4_144146_read_qualsA csv documenting the median qualities of each position for each tag group in gDNA 10^-4 sample, replicate1. The header information is in the README.txt file.KRAS_10-4_145147_read_qualsA csv documenting the median qualities of each position for each tag group in gDNA 10^-4 sample, replicate2. The header information is in the README.txt file.KRAS_10-3_144147_read_qualsA csv documenting the median qualities of each position for each tag group in gDNA 10^-3 sample, replicate1. The header information is in the README.txt file.KRAS_10-3_145148_read_qualsA csv documenting the median qualities of each position for each tag group in gDNA 10^-3 sample, replicate2. The header information is in the README.txt file.KRAS_10-2_144148_read_qualsA csv documenting the median qualities of each position for each tag group in gDNA 10^-2 sample, replicate1. The header information is in the README.txt file.KRAS_10-2_145149_read_qualsA csv documenting the median qualities of each position for each tag group in gDNA 10^-2 sample, replicate2. The header information is in the README.txt file.KRAS_10-1_144149_read_qualsA csv documenting the median qualities of each position for each tag group in gDNA 10^-1 sample, replicate1. The header information is in the README.txt file.KRAS_10-1_145150_read_qualsA csv documenting the median qualities of each position for each tag group in gDNA 10^-1 sample, replicate2. The header information is in the README.txt file.KRAS_MUT_144150_read_qualsA csv documenting the median qualities of each position for each tag group in gDNA mutant only sample, replicate1. The header information is in the README.txt file.KRAS_MUT_145151_read_qualsA csv documenting the median qualities of each position for each tag group in gDNA mutant only sample, replicate2. The header information is in the README.txt file.KRAS_WT_144144_filtered.csvA gzipped csv documenting the tag, read sequence, and associated qualities of all reads matching the majority read length (documented in read lengths csv) of gDNA WT only sample, replicate1. The header information is in the README.txt file.KRAS_WT_145145_filtered.csvA gzipped csv documenting the tag, read sequence, and associated qualities of all reads matching the majority read length (documented in read lengths csv) of gDNA WT only sample, replicate2. The header information is in the README.txt file.KRAS_10-5_144145_filtered.csvA gzipped csv documenting the tag, read sequence, and associated qualities of all reads matching the majority read length (documented in read lengths csv) of gDNA 10^-5 sample, replicate1. The header information is in the README.txt file.KRAS_10-5_145146_filtered.csvA gzipped csv documenting the tag, read sequence, and associated qualities of all reads matching the majority read length (documented in read lengths csv) of gDNA 10^-5 sample, replicate2. The header information is in the README.txt file.KRAS_10-4_144146_filtered.csvA gzipped csv documenting the tag, read sequence, and associated qualities of all reads matching the majority read length (documented in read lengths csv) of gDNA 10^-4 sample, replicate1. The header information is in the README.txt file.KRAS_10-4_145147_filtered.csvA gzipped csv documenting the tag, read sequence, and associated qualities of all reads matching the majority read length (documented in read lengths csv) of gDNA 10^-4 sample, replicate2. The header information is in the README.txt file.KRAS_10-3_144147_filtered.csvA gzipped csv documenting the tag, read sequence, and associated qualities of all reads matching the majority read length (documented in read lengths csv) of gDNA 10^-3 sample, replicate1. The header information is in the README.txt file.KRAS_10-3_145148_filtered.csvA gzipped csv documenting the tag, read sequence, and associated qualities of all reads matching the majority read length (documented in read lengths csv) of gDNA 10^-3 sample, replicate2. The header information is in the README.txt file.KRAS_10-2_144148_filtered.csvA gzipped csv documenting the tag, read sequence, and associated qualities of all reads matching the majority read length (documented in read lengths csv) of gDNA 10^-2 sample, replicate1. The header information is in the README.txt file.KRAS_10-2_145149_filtered.csvA gzipped csv documenting the tag, read sequence, and associated qualities of all reads matching the majority read length (documented in read lengths csv) of gDNA 10^-2 sample, replicate2. The header information is in the README.txt file.KRAS_10-1_144149_filtered.csvA gzipped csv documenting the tag, read sequence, and associated qualities of all reads matching the majority read length (documented in read lengths csv) of gDNA 10^-1 sample, replicate1. The header information is in the README.txt file.KRAS_10-1_145150_filtered.csvA gzipped csv documenting the tag, read sequence, and associated qualities of all reads matching the majority read length (documented in read lengths csv) of gDNA 10^-1 sample, replicate2. The header information is in the README.txt file.KRAS_MUT_144150_filtered.csvA gzipped csv documenting the tag, read sequence, and associated qualities of all reads matching the majority read length (documented in read lengths csv) of gDNA mutant only sample, replicate1. The header information is in the README.txt file.KRAS_MUT_145151_filtered.csvA gzipped csv documenting the tag, read sequence, and associated qualities of all reads matching the majority read length (documented in read lengths csv) of gDNA mutant only sample, replicate2. The header information is in the README.txt file.PCR_stds_error_posA csv file documenting the observed error at targeted position (position 10 of reference) for each PCR-based sample investigated.Kras_WT_132132_nuc_countsA csv file containing the nucleotide counts for each position of PCR-based WT sample, replicate 1.Kras_WT_132133_nuc_countsA csv file containing the nucleotide counts for each position of PCR-based WT sample, replicate 2.Kras_WT_132134_nuc_countsA csv file containing the nucleotide counts for each position of PCR-based WT sample, replicate 3.Kras_10-5_132135_nuc_countsA csv file containing the nucleotide counts for each position of PCR-based 10^-5 sample, replicate 1.Kras_10-5_132136_nuc_countsA csv file containing the nucleotide counts for each position of PCR-based 10^-5 sample, replicate 2.Kras_10-5_132149_nuc_countsA csv file containing the nucleotide counts for each position of PCR-based 10^-5 sample, replicate 3.Kras_10-4_132137_nuc_countsA csv file containing the nucleotide counts for each position of PCR-based 10^-4 sample, replicate 1.Kras_10-4_132138_nuc_countsA csv file containing the nucleotide counts for each position of PCR-based 10^-4 sample, replicate 2.Kras_10-4_132139_nuc_countsA csv file containing the nucleotide counts for each position of PCR-based 10^-4 sample, replicate 3.Kras_10-3_132140_nuc_countsA csv file containing the nucleotide counts for each position of PCR-based 10^-3 sample, replicate 1.Kras_10-3_132141_nuc_countsA csv file containing the nucleotide counts for each position of PCR-based 10^-3 sample, replicate 2.Kras_10-3_132142_nuc_countsA csv file containing the nucleotide counts for each position of PCR-based 10^-3 sample, replicate 3.Kras_10-2_132143_nuc_countsA csv file containing the nucleotide counts for each position of PCR-based 10^-2 sample, replicate 1.Kras_10-2_132144_nuc_countsA csv file containing the nucleotide counts for each position of PCR-based 10^-2 sample, replicate 2.Kras_10-2_132145_nuc_countsA csv file containing the nucleotide counts for each position of PCR-based 10^-2 sample, replicate 3.Kras_10-1_132146_nuc_countsA csv file containing the nucleotide counts for each position of PCR-based 10^-1 sample, replicate 1.Kras_10-1_132147_nuc_countsA csv file containing the nucleotide counts for each position of PCR-based 10^-1 sample, replicate 2.Kras_10-1_132148_nuc_countsA csv file containing the nucleotide counts for each position of PCR-based 10^-1 sample, replicate 3.Kras_MUT_132150_nuc_countsA csv file containing the nucleotide counts for each position of PCR-based mutant only sample, replicate 1.Kras_MUT_132151_nuc_countsA csv file containing the nucleotide counts for each position of PCR-based mutant only sample, replicate 2.Kras_MUT_132152_nuc_countsA csv file containing the nucleotide counts for each position of PCR-based mutant only sample, replicate 3.PCR_all_group_size_histoA csv file containing data describing the number of groups with specified number of reads in the group for the PCR-based samples. The first column indicates the number of reads, the remaining column headers are the sample names, and the list of numbers under each name is the number of groups of specified read depth.PCR_all_group_read_lens_histoA csv file of the read lengths for each PCR-based sample, with length as the row name and the number of reads with specified length for each sample across the columns. Column headers are the names of the samples.Kras_WT_132132_read_qualsA csv documenting the median qualities of each position for each tag group in PCR-based WT sample, replicate1. The header information is in the README.txt file.Kras_WT_132133_read_qualsA csv documenting the median qualities of each position for each tag group in PCR-based WT sample, replicate2. The header information is in the README.txt file.Kras_WT_132134_read_qualsA csv documenting the median qualities of each position for each tag group in PCR-based WT sample, replicate3. The header information is in the README.txt file.Kras_10-5_132135_read_qualsA csv documenting the median qualities of each position for each tag group in PCR-based 10^-5 sample, replicate1. The header information is in the README.txt file.Kras_10-5_132136_read_qualsA csv documenting the median qualities of each position for each tag group in PCR-based 10^-5 sample, replicate2. The header information is in the README.txt file.Kras_10-5_132149_read_qualsA csv documenting the median qualities of each position for each tag group in PCR-based 10^-5 sample, replicate3. The header information is in the README.txt file.Kras_10-4_132137_read_qualsA csv documenting the median qualities of each position for each tag group in PCR-based 10^-4 sample, replicate1. The header information is in the README.txt file.Kras_10-4_132138_read_qualsA csv documenting the median qualities of each position for each tag group in PCR-based 10^-4 sample, replicate2. The header information is in the README.txt file.Kras_10-4_132139_read_qualsA csv documenting the median qualities of each position for each tag group in PCR-based 10^-4 sample, replicate3. The header information is in the README.txt file.Kras_10-3_132140_read_qualsA csv documenting the median qualities of each position for each tag group in PCR-based 10^-3 sample, replicate1. The header information is in the README.txt file.Kras_10-3_132141_read_qualsA csv documenting the median qualities of each position for each tag group in PCR-based 10^-3 sample, replicate2. The header information is in the README.txt file.Kras_10-3_132142_read_qualsA csv documenting the median qualities of each position for each tag group in PCR-based 10^-3 sample, replicate3. The header information is in the README.txt file.Kras_10-2_132143_read_qualsA csv documenting the median qualities of each position for each tag group in PCR-based 10^-2 sample, replicate1. The header information is in the README.txt file.Kras_10-2_132144_read_qualsA csv documenting the median qualities of each position for each tag group in PCR-based 10^-2 sample, replicate2. The header information is in the README.txt file.Kras_10-2_132145_read_qualsA csv documenting the median qualities of each position for each tag group in PCR-based 10^-2 sample, replicate3. The header information is in the README.txt file.Kras_10-1_132146_read_qualsA csv documenting the median qualities of each position for each tag group in PCR-based 10^-1 sample, replicate1. The header information is in the README.txt file.Kras_10-1_132147_read_qualsA csv documenting the median qualities of each position for each tag group in PCR-based 10^-1 sample, replicate2. The header information is in the README.txt file.Kras_10-1_132148_read_qualsA csv documenting the median qualities of each position for each tag group in PCR-based 10^-1 sample, replicate3. The header information is in the README.txt file.Kras_MUT_132150_read_qualsA csv documenting the median qualities of each position for each tag group in PCR-based mutant only sample, replicate1. The header information is in the README.txt file.Kras_MUT_132151_read_qualsA csv documenting the median qualities of each position for each tag group in PCR-based mutant only sample, replicate2. The header information is in the README.txt file.Kras_MUT_132152_read_qualsA csv documenting the median qualities of each position for each tag group in PCR-based mutant only sample, replicate3. The header information is in the README.txt file.Kras_WT_132132_filtered.csvA gzipped csv documenting the tag, read sequence, and associated qualities of all reads matching the majority read length (documented in read lengths csv) of PCR-based WT only sample, replicate1. The header information is in the README.txt file.Kras_WT_132133_filtered.csvA gzipped csv documenting the tag, read sequence, and associated qualities of all reads matching the majority read length (documented in read lengths csv) of PCR-based WT only sample, replicate2. The header information is in the README.txt file.Kras_WT_132134_filtered.csvA gzipped csv documenting the tag, read sequence, and associated qualities of all reads matching the majority read length (documented in read lengths csv) of PCR-based WT only sample, replicate3. The header information is in the README.txt file.Kras_10-5_132135_filtered.csvA gzipped csv documenting the tag, read sequence, and associated qualities of all reads matching the majority read length (documented in read lengths csv) of PCR-based 10^-5 only sample, replicate1. The header information is in the README.txt file.Kras_10-5_132136_filtered.csvA gzipped csv documenting the tag, read sequence, and associated qualities of all reads matching the majority read length (documented in read lengths csv) of PCR-based 10^-5 only sample, replicate2. The header information is in the README.txt file.Kras_10-5_132149_filtered.csvA gzipped csv documenting the tag, read sequence, and associated qualities of all reads matching the majority read length (documented in read lengths csv) of PCR-based 10^-5 only sample, replicate3. The header information is in the README.txt file.Kras_10-4_132137_filtered.csvA gzipped csv documenting the tag, read sequence, and associated qualities of all reads matching the majority read length (documented in read lengths csv) of PCR-based 10^-4 only sample, replicate1. The header information is in the README.txt file.Kras_10-4_132138_filtered.csvA gzipped csv documenting the tag, read sequence, and associated qualities of all reads matching the majority read length (documented in read lengths csv) of PCR-based 10^-4 only sample, replicate2. The header information is in the README.txt file.Kras_10-4_132139_filtered.csvA gzipped csv documenting the tag, read sequence, and associated qualities of all reads matching the majority read length (documented in read lengths csv) of PCR-based 10^-4 only sample, replicate3. The header information is in the README.txt file.Kras_10-3_132140_filtered.csvA gzipped csv documenting the tag, read sequence, and associated qualities of all reads matching the majority read length (documented in read lengths csv) of PCR-based 10^-3 only sample, replicate1. The header information is in the README.txt file.Kras_10-3_132141_filtered.csvA gzipped csv documenting the tag, read sequence, and associated qualities of all reads matching the majority read length (documented in read lengths csv) of PCR-based 10^-3 only sample, replicate2. The header information is in the README.txt file.Kras_10-3_132142_filtered.csvA gzipped csv documenting the tag, read sequence, and associated qualities of all reads matching the majority read length (documented in read lengths csv) of PCR-based 10^-3 only sample, replicate3. The header information is in the README.txt file.Kras_10-2_132143_filtered.csvA gzipped csv documenting the tag, read sequence, and associated qualities of all reads matching the majority read length (documented in read lengths csv) of PCR-based 10^-2 only sample, replicate1. The header information is in the README.txt file.Kras_10-2_132144_filtered.csvA gzipped csv documenting the tag, read sequence, and associated qualities of all reads matching the majority read length (documented in read lengths csv) of PCR-based 10^-2 only sample, replicate2. The header information is in the README.txt file.Kras_10-2_132145_filtered.csvA gzipped csv documenting the tag, read sequence, and associated qualities of all reads matching the majority read length (documented in read lengths csv) of PCR-based 10^-2 only sample, replicate3. The header information is in the README.txt file.Kras_10-1_132146_filtered.csvA gzipped csv documenting the tag, read sequence, and associated qualities of all reads matching the majority read length (documented in read lengths csv) of PCR-based 10^-1 only sample, replicate1. The header information is in the README.txt file.Kras_10-1_132147_filtered.csvA gzipped csv documenting the tag, read sequence, and associated qualities of all reads matching the majority read length (documented in read lengths csv) of PCR-based 10^-1 only sample, replicate2. The header information is in the README.txt file.Kras_10-1_132148_filtered.csvA gzipped csv documenting the tag, read sequence, and associated qualities of all reads matching the majority read length (documented in read lengths csv) of PCR-based 10^-1 only sample, replicate3. The header information is in the README.txt file.Kras_MUT_132150_filtered.csvA gzipped csv documenting the tag, read sequence, and associated qualities of all reads matching the majority read length (documented in read lengths csv) of PCR-based mutant only only sample, replicate1. The header information is in the README.txt file.Kras_MUT_132151_filtered.csvA gzipped csv documenting the tag, read sequence, and associated qualities of all reads matching the majority read length (documented in read lengths csv) of PCR-based mutant only only sample, replicate2. The header information is in the README.txt file.Kras_MUT_132152_filtered.csvA gzipped csv documenting the tag, read sequence, and associated qualities of all reads matching the majority read length (documented in read lengths csv) of PCR-based mutant only only sample, replicate3. The header information is in the README.txt file.