DOI: 10.5061/dryad.xpnvx0kft
title: CarcSeq measurement of lung cancer driver mutations predicts mouse strain- and sex-related incidence of spontaneous lung neoplasia

description: The data contained herein are the fastq files collected for individual
 mouse lung DNA samples. Ten samples each of male and female, B6C3F1 and
 CD-1 mouse lung DNA were analyzed using the CarcSeq method for
 error-corrected next-generation sequencing as previously
 described [Harris, KL, Walia, V, Gong, B, et al. Quantification of cancer
 driver mutations in human breast and lung DNA using targeted,
 error-corrected CarcSeq. Environ Mol Mutagen. 2020; 61: 872– 889.
 https://doi.org/10.1002/em.22409 and Karen L McKim,
 Meagan B Myers, Kelly L Harris, Binsheng Gong, Joshua Xu, Barbara L
 Parsons, CarcSeq Measurement of Rat Mammary Cancer Driver Mutations and
 Relation to Spontaneous Mammary Neoplasia, Toxicological Sciences, 2021;,
 kfab040, https://doi.org/10.1093/toxsci/kfab040]. A readme text
 file provides the key to which sample numbers correspond with which mouse
 strain and sex. All the fastq files within a given folder were used to
 construct single strand consensus sequences, which were then subject to
 downstream analyses as reported in the associated publication.
description: From each mouse sample (40, 10 each male and female, B6C3F1 and
 CD-1 mice), first-round PCR was conducted to generate14 amplicons of mouse
 homologs of human sequences encompassing hotspot cancer driver mutations
 (CDMs). The primers used were constructed with 9 bases of degenerate
 sequence at their 5' ends, resulting in 18 basepairs of sequence that
 served as a unique molecular identifier during error correction. The
 amplicons from the same mouse sample were combined and the Illumina®
 TruSeq® ChIP Sample Preparation Kit (Illumina, San Diego, CA) was used to
 construct a library, suitable for Illumina sequencing. The method for
 library preparation was modified from that provided by the manufacturer in
 that DNAs recovered from the gel purification step were diluted serially
 and numbers of PCR-amplifiable molecules in the dilutions were determined
 by digital droplet PCR (ddPCR), using the ddPCR Library Quantification Kit
 for Illumina (BioRad, Hercules, CA) and the QX200 Droplet Digital PCR
 System (Bio-Rad). Based on this ddPCR quantification, 1.5 x
 10<sup>6</sup> molecules were added to the final PCR described
 in the Illumina® TruSeq® ChIP kit protocol. Mouse lung DNA libraries were
 pooled, denatured, and diluted according to the Illumina® TruSeq® Library
 Prep Pooling Guide. Specifically, libraries were diluted to 2 nM, combined
 with 2 nM of PhiX (Illumina), denatured and diluted, then loaded onto an
 Illumina NextSeq500 for cluster generation and 151-cycle, paired-end
 sequencing using NSQ 500/550 Mid or High Output KT (300 CYS) reagents
 using a six bp index read. We employed methods reported by Kennedy et al.
 [Kennedy, S.R, Schmitt, M.W, Fox, E.J, Kohrn, B.F, Salk, J.J, Ahn, E.H,
 Prindle, M.J, Kuong, K.J, Shen, J.-C, Risques, R.-A & Loeb, L.A
 (2014). Detecting ultralow-frequency mutations by Duplex
 Sequencing.<br> Nature Protocols, 9(11), 2586–2606] for error
 correction, except recovering single-strand consensus sequences (SSCSs)
 for use in all downstream analyses. Library preparation and sequencing
 were repeated if 100,000 SSCSs were not recovered, in which case fastq
 files from multiple library preparations were combined for SSCS
 construction.
description: A ReadMe text file was uploaded that provides a sample key for the data in
 the folders.