Reducing mitochondrial reads in ATAC-seq using CRISPR/Cas9

Lindsey Montefiori,Gregory Crawford,Yoav Gilad,Zijie Zhang,Liana Hernandez,Carole Ober,Marcelo Nobrega,Noboru Jo Sakabe

doi:10.1038/s41598-017-02547-w

Lindsey Montefiori, Gregory Crawford + Show 6 more

Open Access

https://doi.org/10.1038/s41598-017-02547-w

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Journal: Scientific Reports	Publication Date: May 26, 2017
Citations: 51	License type: open-access

Affiliation: University of Chicago, Duke University

Abstract

ATAC-seq is a high-throughput sequencing technique that identifies open chromatin. Depending on the cell type, ATAC-seq samples may contain ~20–80% of mitochondrial sequencing reads. As the regions of open chromatin of interest are usually located in the nuclear genome, mitochondrial reads are typically discarded from the analysis. We tested two approaches to decrease wasted sequencing in ATAC-seq libraries generated from lymphoblastoid cell lines: targeted cleavage of mitochondrial DNA fragments using CRISPR technology and removal of detergent from the cell lysis buffer. We analyzed the effects of these treatments on the number of usable (unique, non-mitochondrial) reads and the number and quality of peaks called, including peaks identified in enhancers and transcription start sites. Both treatments resulted in considerable reduction of mitochondrial reads (1.7 and 3-fold, respectively). The removal of detergent, however, resulted in increased background and fewer peaks. The highest number of peaks and highest quality data was obtained by preparing samples with the original ATAC-seq protocol (using detergent) and treating them with CRISPR. This strategy reduced the amount of sequencing required to call a high number of peaks, which could lead to cost reduction when performing ATAC-seq on large numbers of samples and in cell types that contain a large amount of mitochondria.

Highlights

ATAC-seq aims at identifying DNA sequences located in open chromatin, i.e., genomic regions whose chromatin is not densely packaged and that can be more accessed by proteins than closed chromatin
Whereas the removal of detergent from the lysis buffer had the largest effect in reducing mitochondrial reads, it resulted in decreased quality of the ATAC-seq libraries, as measured by the number of peaks called at a given sequencing depth, the total number of reads in peaks, and the fraction of transcription start sites (TSS’s) and enhancers identified
Treating the samples before PCR amplification might result in lower fractions of mitochondria, we chose the conservative approach of treating larger amounts of DNA to reduce technical variability

Summary

Introduction

ATAC-seq aims at identifying DNA sequences located in open chromatin, i.e., genomic regions whose chromatin is not densely packaged and that can be more accessed by proteins than closed chromatin. To analyze the effect of this approach on the quality of the data, we designed 100 gRNAs targeting the human mitochondrial chromosome every ~250 base pairs and treated lymphoblastoid cell line (LCL) ATAC-seq sequencing libraries with these gRNAs and Cas[9] enzyme[4], hereafter referred to as anti-mt CRISPR. We compared this method to a modified ATAC-seq protocol that aims at reducing mitochondrial reads by removing detergent from the cell lysis step, which is believed to prevent lysis of the mitochondrial membrane[5]. The gRNAs can be generated from template DNA oligos indefinitely and shared www.nature.com/scientificreports/

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