Publications by authors named "Brendan F Kohrn"

8 Publications

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PolyG-DS: An ultrasensitive polyguanine tract-profiling method to detect clonal expansions and trace cell lineage.

Proc Natl Acad Sci U S A 2021 Aug;118(31)

Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA 98195;

Polyguanine tracts (PolyGs) are short guanine homopolymer repeats that are prone to accumulating mutations when cells divide. This feature makes them especially suitable for cell lineage tracing, which has been exploited to detect and characterize precancerous and cancerous somatic evolution. PolyG genotyping, however, is challenging because of the inherent biochemical difficulties in amplifying and sequencing repetitive regions. To overcome this limitation, we developed PolyG-DS, a next-generation sequencing (NGS) method that combines the error-correction capabilities of duplex sequencing (DS) with enrichment of PolyG loci using CRISPR-Cas9-targeted genomic fragmentation. PolyG-DS markedly reduces technical artifacts by comparing the sequences derived from the complementary strands of each original DNA molecule. We demonstrate that PolyG-DS genotyping is accurate, reproducible, and highly sensitive, enabling the detection of low-frequency alleles (<0.01) in spike-in samples using a panel of only 19 PolyG markers. PolyG-DS replicated prior results based on PolyG fragment length analysis by capillary electrophoresis, and exhibited higher sensitivity for identifying clonal expansions in the nondysplastic colon of patients with ulcerative colitis. We illustrate the utility of this method for resolving the phylogenetic relationship among precancerous lesions in ulcerative colitis and for tracing the metastatic dissemination of ovarian cancer. PolyG-DS enables the study of tumor evolution without prior knowledge of tumor driver mutations and provides a tool to perform cost-effective and easily scalable ultra-accurate NGS-based PolyG genotyping for multiple applications in biology, genetics, and cancer research.
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http://dx.doi.org/10.1073/pnas.2023373118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8346827PMC
August 2021

Extensive subclonal mutational diversity in human colorectal cancer and its significance.

Proc Natl Acad Sci U S A 2019 Dec 5. Epub 2019 Dec 5.

Department of Oncology, Georgetown University Medical Center, Washington, DC 20007.

Human colorectal cancers (CRCs) contain both clonal and subclonal mutations. Clonal driver mutations are positively selected, present in most cells, and drive malignant progression. Subclonal mutations are randomly dispersed throughout the genome, providing a vast reservoir of mutant cells that can expand, repopulate the tumor, and result in the rapid emergence of resistance, as well as being a major contributor to tumor heterogeneity. Here, we apply duplex sequencing (DS) methodology to quantify subclonal mutations in CRC tumor with unprecedented depth (10) and accuracy (<10). We measured mutation frequencies in genes encoding replicative DNA polymerases and in genes frequently mutated in CRC, and found an unexpectedly high effective mutation rate, 7.1 × 10 The curve of subclonal mutation accumulation as a function of sequencing depth, using DNA obtained from 5 different tumors, is in accord with a neutral model of tumor evolution. We present a theoretical approach to model neutral evolution independent of the infinite-sites assumption (which states that a particular mutation arises only in one tumor cell at any given time). Our analysis indicates that the infinite-sites assumption is not applicable once the number of tumor cells exceeds the reciprocal of the mutation rate, a circumstance relevant to even the smallest clinically diagnosable tumor. Our methods allow accurate estimation of the total mutation burden in clinical cancers. Our results indicate that no DNA locus is wild type in every malignant cell within a tumor at the time of diagnosis (probability of all cells being wild type, 10).
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http://dx.doi.org/10.1073/pnas.1910301116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6936702PMC
December 2019

A high-resolution landscape of mutations in the super-enhancer in normal human B cells.

Proc Natl Acad Sci U S A 2019 12 20;116(49):24779-24785. Epub 2019 Nov 20.

Department of Pathology, University of Washington, Seattle, WA 98195;

The super-enhancers (SEs) of lineage-specific genes in B cells are off-target sites of somatic hypermutation. However, the inability to detect sufficient numbers of mutations in normal human B cells has precluded the generation of a high-resolution mutational landscape of SEs. Here we captured and sequenced 12 B cell SEs at single-nucleotide resolution from 10 healthy individuals across diverse ethnicities. We detected a total of approximately 9,000 subclonal mutations (allele frequencies <0.1%); of these, approximately 8,000 are present in the SE alone. Within the SE, we identified 3 regions of clustered mutations in which the mutation frequency is ∼7 × 10 Mutational spectra show a predominance of C > T/G > A and A > G/T > C substitutions, consistent with the activities of activation-induced-cytidine deaminase (AID) and the A-T mutator, DNA polymerase η, respectively, in mutagenesis in normal B cells. Analyses of mutational signatures further corroborate the participation of these factors in this process. Single base substitution signatures SBS85, SBS37, and SBS39 were found in the SE. While SBS85 is a denoted signature of AID in lymphoid cells, the etiologies of SBS37 and SBS39 are unknown. Our analysis suggests the contribution of error-prone DNA polymerases to the latter signatures. The high-resolution mutation landscape has enabled accurate profiling of subclonal mutations in B cell SEs in normal individuals. By virtue of the fact that subclonal SE mutations are clonally expanded in B cell lymphomas, our studies also offer the potential for early detection of neoplastic alterations.
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http://dx.doi.org/10.1073/pnas.1914163116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6900602PMC
December 2019

Targeted genome fragmentation with CRISPR/Cas9 enables fast and efficient enrichment of small genomic regions and ultra-accurate sequencing with low DNA input (CRISPR-DS).

Genome Res 2018 10 19;28(10):1589-1599. Epub 2018 Sep 19.

Department of Pathology, University of Washington, Seattle, Washington 98195, USA.

Next-generation sequencing methods suffer from low recovery, uneven coverage, and false mutations. DNA fragmentation by sonication is a major contributor to these problems because it produces randomly sized fragments, PCR amplification bias, and end artifacts. In addition, oligonucleotide-based hybridization capture, a common target enrichment method, has limited efficiency for small genomic regions, contributing to low recovery. This becomes a critical problem in clinical applications, which value cost-effective approaches focused on the sequencing of small gene panels. To address these issues, we developed a targeted genome fragmentation approach based on CRISPR/Cas9 digestion that produces DNA fragments of similar length. These fragments can be enriched by a simple size selection, resulting in targeted enrichment of up to approximately 49,000-fold. Additionally, homogenous length fragments significantly reduce PCR amplification bias and maximize read usability. We combined this novel target enrichment approach with Duplex Sequencing, which uses double-strand molecular tagging to correct for sequencing errors. The approach, termed CRISPR-DS, enables efficient target enrichment of small genomic regions, even coverage, ultra-accurate sequencing, and reduced DNA input. As proof of principle, we applied CRISPR-DS to the sequencing of the exonic regions of and performed side-by-side comparisons with standard Duplex Sequencing. CRISPR-DS detected previously reported pathogenic mutations present as low as 0.1% in peritoneal fluid of women with ovarian cancer, while using 10- to 100-fold less DNA than standard Duplex Sequencing. Whether used as standalone enrichment or coupled with high-accuracy sequencing methods, CRISPR-based fragmentation offers a simple solution for fast and efficient small target enrichment.
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http://dx.doi.org/10.1101/gr.235291.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6169890PMC
October 2018

An efficient pipeline to generate data for studies in plastid population genomics and phylogeography.

Appl Plant Sci 2017 Nov 14;5(11). Epub 2017 Nov 14.

Department of Biology, Portland State University, 1719 SW 10th Avenue, Portland, Oregon 97201 USA.

Premise Of The Study: Seed dispersal contributes to gene flow and is responsible for colonization of new sites and range expansion. Sequencing chloroplast haplotypes offers a way to estimate contributions of seed dispersal to population genetic structure and enables studies of population history. Whole-genome sequencing is expensive, but resources can be conserved by pooling samples. Unfortunately, haplotype associations among single-nucleotide polymorphisms (SNPs) are lost in pooled samples, and treating SNP allele frequencies as independent markers provides biased estimates of genetic structure.

Methods: We developed sampling methodologies and an application, CallHap, that uses a least-squares algorithm to evaluate the fit between observed and predicted SNP allele frequencies from pooled samples based on haplotype network phylogeny structure, thus enabling pooling for chloroplast sequencing for large-scale studies of chloroplast genomic variation. This method was tested using artificially constructed test networks and pools, and pooled samples of (California goldfields) from southern Oregon, USA.

Results: CallHap reliably recovered network topologies and haplotype frequencies from pooled samples.

Discussion: The CallHap pipeline allows for the efficient use of resources for estimation of genetic structure for studies using nonrecombining haplotypes such as intraspecific variation in chloroplast, mitochondrial, bacterial, or viral DNA.
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http://dx.doi.org/10.3732/apps.1700053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5703179PMC
November 2017

Small unmanned aerial vehicles (micro-UAVs, drones) in plant ecology.

Appl Plant Sci 2016 Sep 19;4(9). Epub 2016 Sep 19.

Department of Biology, Portland State University, P.O. Box 751, Portland, Oregon 97207 USA.

Premise Of The Study: Low-elevation surveys with small aerial drones (micro-unmanned aerial vehicles [UAVs]) may be used for a wide variety of applications in plant ecology, including mapping vegetation over small- to medium-sized regions. We provide an overview of methods and procedures for conducting surveys and illustrate some of these applications.

Methods: Aerial images were obtained by flying a small drone along transects over the area of interest. Images were used to create a composite image (orthomosaic) and a digital surface model (DSM). Vegetation classification was conducted manually and using an automated routine. Coverage of an individual species was estimated from aerial images.

Results: We created a vegetation map for the entire region from the orthomosaic and DSM, and mapped the density of one species. Comparison of our manual and automated habitat classification confirmed that our mapping methods were accurate. A species with high contrast to the background matrix allowed adequate estimate of its coverage.

Discussion: The example surveys demonstrate that small aerial drones are capable of gathering large amounts of information on the distribution of vegetation and individual species with minimal impact to sensitive habitats. Low-elevation aerial surveys have potential for a wide range of applications in plant ecology.
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http://dx.doi.org/10.3732/apps.1600041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5033362PMC
September 2016

Detection of Ultra-Rare Mitochondrial Mutations in Breast Stem Cells by Duplex Sequencing.

PLoS One 2015 25;10(8):e0136216. Epub 2015 Aug 25.

Department of Pathology, University of Washington, Seattle, Washington, United States of America; Department of Biochemistry, University of Washington, Seattle, Washington, United States of America.

Long-lived adult stem cells could accumulate non-repaired DNA damage or mutations that increase the risk of tumor formation. To date, studies on mutations in stem cells have concentrated on clonal (homoplasmic) mutations and have not focused on rarely occurring stochastic mutations that may accumulate during stem cell dormancy. A major challenge in investigating these rare mutations is that conventional next generation sequencing (NGS) methods have high error rates. We have established a new method termed Duplex Sequencing (DS), which detects mutations with unprecedented accuracy. We present a comprehensive analysis of mitochondrial DNA mutations in human breast normal stem cells and non-stem cells using DS. The vast majority of mutations occur at low frequency and are not detectable by NGS. The most prevalent point mutation types are the C>T/G>A and A>G/T>C transitions. The mutations exhibit a strand bias with higher prevalence of G>A, T>C, and A>C mutations on the light strand of the mitochondrial genome. The overall rare mutation frequency is significantly lower in stem cells than in the corresponding non-stem cells. We have identified common and unique non-homoplasmic mutations between non-stem and stem cells that include new mutations which have not been reported previously. Four mutations found within the MT-ND5 gene (m.12684G>A, m.12705C>T, m.13095T>C, m.13105A>G) are present in all groups of stem and non-stem cells. Two mutations (m.8567T>C, m.10547C>G) are found only in non-stem cells. This first genome-wide analysis of mitochondrial DNA mutations may aid in characterizing human breast normal epithelial cells and serve as a reference for cancer stem cell mutation profiles.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0136216PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4549069PMC
May 2016

Detecting ultralow-frequency mutations by Duplex Sequencing.

Nat Protoc 2014 Nov 9;9(11):2586-606. Epub 2014 Oct 9.

1] Department of Pathology, University of Washington, Seattle, USA. [2] Department of Biochemistry, University of Washington, Seattle, USA.

Duplex Sequencing (DS) is a next-generation sequencing methodology capable of detecting a single mutation among >1 × 10(7) wild-type nucleotides, thereby enabling the study of heterogeneous populations and very-low-frequency genetic alterations. DS can be applied to any double-stranded DNA sample, but it is ideal for small genomic regions of <1 Mb in size. The method relies on the ligation of sequencing adapters harboring random yet complementary double-stranded nucleotide sequences to the sample DNA of interest. Individually labeled strands are then PCR-amplified, creating sequence 'families' that share a common tag sequence derived from the two original complementary strands. Mutations are scored only if the variant is present in the PCR families arising from both of the two DNA strands. Here we provide a detailed protocol for efficient DS adapter synthesis, library preparation and target enrichment, as well as an overview of the data analysis workflow. The protocol typically takes 1-3 d.
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http://dx.doi.org/10.1038/nprot.2014.170DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4271547PMC
November 2014
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