XR-seq Bioinformatics Pipeline

Genome-wide mapping of nucleotide excision repair with XR-seq

Nucleotide excision repair is a versatile mechanism to repair a variety of bulky DNA adducts. We developed eXcision Repair sequencing (XR-seq) to study nucleotide excision repair of several bulky DNA adducts in humans, mice, Arabidopsis thaliana, yeast, and Escherichia coli. In this protocol, the excised oligomers, generated in the nucleotide excision repair reaction are isolated by cell lysis and fractionation, followed by immunoprecipitation with damage- or repair factor-specific antibodies from the non-chromatin fraction. The single-stranded excised oligomers are ligated to adaptors and re-immunoprecipitated with damage-specific antibodies. The DNA damage in the excised oligomers is then reversed by enzymatic or chemical reactions before being converted into a sequencing library by PCR amplification. Alternatively, the excised oligomers containing DNA damage, especially those containing irreversible DNA damage such as benzo[a]pyrene-induced DNA adducts, can be converted to a dsDNA form by using appropriate translesion DNA synthesis (TLS) polymerases and amplified by PCR. Current genome-wide approaches to study repair measure the loss of damage signal with time, which limits the resolution. In contrast, an advantage of XR-seq is that the repair signal is directly detected above a background of zero. An XR-seq library using the protocol described here can be obtained in 7 – 9 d.

About release

This release is published with the aim of providing the bioinformatic pipeline to perform the preliminary XR-seq analysis mentioned in our associated manuscript.

XR-seq Protocol - Bioinformatics Pipeline

Genome-wide mapping of nucleotide excision repair with XR-seq

Nucleotide excision repair is a versatile mechanism to repair a variety of bulky DNA adducts. We developed eXcision Repair sequencing (XR-seq) to study nucleotide excision repair of several bulky DNA adducts in humans, mice, Arabidopsis thaliana, yeast, and Escherichia coli. In this protocol the excised oligomers, generated in the nucleotide excision repair reaction are isolated by cell lysis and fractionation, followed by immunoprecipitation with damage- or repair factor-specific antibodies from the non-chromatin fraction. The single-stranded excised oligomers are ligated to adaptors and re-immunoprecipitated with damage-specific antibodies. The DNA damage in the excised oligomers is then reversed by enzymatic or chemical reactions before being converted into a sequencing library by PCR amplification. Alternatively, the excised oligomers containing DNA damage, especially those containing irreversible DNA damage such as benzo[a]pyrene-induced DNA adducts, can be converted to a dsDNA form by using appropriate translesion DNA synthesis (TLS) polymerases and amplified by PCR. Current genome-wide approaches to study repair measure the loss of damage signal with time, which limits the resolution. In contrast, an advantage of XR-seq is that the repair signal is directly detected above a background of zero. An XR-seq library using the protocol described here can be obtained in 7 – 9 d.

About release

This release is published with the aim of providing the bioinformatic pipeline to perform the preliminary XR-seq analysis mentioned in our associated manuscript.

XR-seq Bioinformatics Pipeline

Genome-wide mapping of nucleotide excision repair with XR-seq

Nucleotide excision repair is a versatile mechanism to repair a variety of bulky DNA adducts. We developed eXcision Repair sequencing (XR-seq) to study nucleotide excision repair of several bulky DNA adducts in humans, mice, Arabidopsis thaliana, yeast, and Escherichia coli. In this protocol, the excised oligomers, generated in the nucleotide excision repair reaction are isolated by cell lysis and fractionation, followed by immunoprecipitation with damage- or repair factor-specific antibodies from the non-chromatin fraction. The single-stranded excised oligomers are ligated to adaptors and re-immunoprecipitated with damage-specific antibodies. The DNA damage in the excised oligomers is then reversed by enzymatic or chemical reactions before being converted into a sequencing library by PCR amplification. Alternatively, the excised oligomers containing DNA damage, especially those containing irreversible DNA damage such as benzo[a]pyrene-induced DNA adducts, can be converted to a dsDNA form by using appropriate translesion DNA synthesis (TLS) polymerases and amplified by PCR. Current genome-wide approaches to study repair measure the loss of damage signal with time, which limits the resolution. In contrast, an advantage of XR-seq is that the repair signal is directly detected above a background of zero. An XR-seq library using the protocol described here can be obtained in 7 – 9 d.

About release

This release is published with the aim of providing the bioinformatic pipeline to perform the preliminary XR-seq analysis mentioned in our associated manuscript.

Arabidopsis

Circadian XR-seq analysis in Arabidopsis

Dependencies:

• bowtie
• bowtie2
• bedtools
• pybedtools

Genomic files should be prepared and their locations should be defined in the referenceGenomePath.py

cd sancarlab_utils/projects/plant

# To run the first sample:
python pipeline.py run -n 1

# OR in slurm cluster:
python pipeline_array -g 0