CIRCLE-seq: Circularization In vitro Reporting of CLeavage Effects by Sequencing

This is a repository for CIRCLE-seq analytical software, which takes sample-specific paired end FASTQ files as input and produces a list of CIRCLE-seq detected off-target cleavage sites as output.

Table of Contents

• Features
• Dependencies
• Getting Set Up
  • Installing Dependencies
  • Download Reference Genome
  • Download and Set Up circleseq
• Usage
  • Quickstart
  • Writing A Manifest File
  • Pipeline Outputs
• Testing the circleseq Package
• Frequently Asked Questions

Features

This package implements a pipeline that takes in reads from the CIRCLE-seq assay and returns detected cleavage sites as output. The individual pipeline steps are:

1. Merge: Merge read1 an read2 for easier mapping to genome.
2. Read Alignment: Merged paired end reads from the assay are aligned to the reference genome using the BWA-MEM algorithm with default parameters (Li. H, 2009).
3. Cleavage Site Identification: Mapped sites are analyzed to determine which represent high-quality cleavage sites.
4. Visualization of Results: Identified on-target and off-target cleavage sites are rendered as a color-coded alignment map for easy analysis of results.

Dependencies

• Python (2.7)
• Reference genome fasta file (Example)
• bwa alignment tool
• samtools alignment format manipulation

Getting Set Up

Install Dependencies

To run circleseq, you must first install all necessary dependencies:

• Python 2.7: If a version does not come bundled with your operating system, we recommend the Anaconda scientific Python package.
• Burrows-Wheeler Aligner (bwa): You can either install bwa with a package manager (e.g. brew on OSX or apt-get on Ubuntu/Debian), or you can download it from the project page and compile it from source.
• Samtools: You can either install samtools with a package manager (e.g. brew or apt-get), or you can download it from the project page and compile it from source.

For both bwa and samtools, make sure you know the path to the respective executables, as they need to be specified in the pipeline manifest file.

Download Reference Genome

The circleseq package requires a reference genome for read mapping. You can use any genome of your choosing, but for all of our testing and original CIRCLE-seq analyses we use hg19 (download). Be sure to (g)unzip the FASTA file before use if it is compressed.

Download and Set Up circleseq

Once all dependencies are installed, there are a few easy steps to download and set up the circleseq package:

1. Download a copy of the circleseq package source code. You can either download and unzip the latest source from the github release page, or you use git to clone the repository by running git clone --recursive https://github.com/tsailabSJ/circleseq.git
2. Install circleseq dependencies by entering the circleseq directory and running pip install -r requirements.txt

Once all circleseq dependencies are installed, you will be ready to start using circleseq.

Usage

Quickstart

Using this tool is simple. After getting set up, create a .yaml manifest file referencing the dependencies and sample .fastq.gz file paths. Then, run python /path/to/circleseq.py all --manifest /path/to/manifest.yaml

Below is an example manifest.yaml file::

reference_genome: /data/joung/genomes/Homo_sapiens_assembly19.fasta
analysis_folder: /data/joung/CIRCLE-Seq/test2

bwa: bwa
samtools: samtools

read_threshold: 4
window_size: 3
mapq_threshold: 50
start_threshold: 1
gap_threshold: 3
mismatch_threshold: 6
merged_analysis: True

samples:
    U2OS_exp1_VEGFA_site_1:
        target: GGGTGGGGGGAGTTTGCTCCNGG
        read1: /data/joung/sequencing_fastq/150902_M01326_0235_000000000-AHLT8/fastq/1_S1_L001_R1_001.fastq.gz
        read2: /data/joung/sequencing_fastq/150902_M01326_0235_000000000-AHLT8/fastq/1_S1_L001_R2_001.fastq.gz
        controlread1: /data/joung/sequencing_fastq/150902_M01326_0235_000000000-AHLT8/fastq/4_S4_L001_R1_001.fastq.gz
        controlread2: /data/joung/sequencing_fastq/150902_M01326_0235_000000000-AHLT8/fastq/4_S4_L001_R2_001.fastq.gz
        description: U2OS_exp1
    U2OS_exp1_EMX1:
        target: GAGTCCGAGCAGAAGAAGAANGG
        read1: /data/joung/sequencing_fastq/150902_M01326_0235_000000000-AHLT8/fastq/2_S2_L001_R1_001.fastq.gz
        read2: /data/joung/sequencing_fastq/150902_M01326_0235_000000000-AHLT8/fastq/2_S2_L001_R2_001.fastq.gz
        controlread1: /data/joung/sequencing_fastq/150902_M01326_0235_000000000-AHLT8/fastq/4_S4_L001_R1_001.fastq.gz
        controlread2: /data/joung/sequencing_fastq/150902_M01326_0235_000000000-AHLT8/fastq/4_S4_L001_R2_001.fastq.gz
        description: U2OS_exp1

Writing A Manifest File

When running the end-to-end analysis functionality of the circleseq package a number of inputs are required. To simplify the formatting of these inputs and to encourage reproducibility, these parameters are inputted into the pipeline via a manifest formatted as a YAML file. YAML files allow easy-to-read specification of key-value pairs. This allows us to easily specify our parameters. The following fields are required in the manifest:

• reference_genome: The absolute path to the reference genome FASTA file.
• output_folder: The absolute path to the folder in which all pipeline outputs will be saved.
• bwa: The absolute path to the bwa executable
• samtools: The absolute path to the samtools executable
• read_threshold: The minimum number of reads at a location for that location to be called as a site. We recommend leaving it to the default value of 4.
• window_size: Size of the sliding window, we recommend leaving it to the default value of 3.
• mapq_threshold: Minimum read mapping quality score. We recommend leaving it to the default value of 50.
• start_threshold: Tolerance for breakpoint location. We recommend leaving it to the default value of 1.
• gap_threshold: Number of tolerated gaps in the fuzzy target search setp. We recommend leaving it to the default value of 3.
• mismatch_threshold: Number of tolerated gaps in the fuzzy target search setp. We recommend leaving it to the default value of 6.
• merged_analysis: Whether or not the paired read merging step should takingTrue
• samples: Lists the samples you wish to analyze and the details for each. Each sample name should be nested under the top level samples key, and each sample detail should be nested under the sample name. See the sample manifest for an example.
  • For each sample, you must provide the following parameters:
    • target: Target sequence for that sample. Accepts degenerate bases.
    • read1: The absolute path to the .FASTQ(.gz) file containing the read1 reads.
    • read2: The absolute path to the .FASTQ(.gz) file containing the read2 reads.
    • controlread1: The absolute path to the .FASTQ(.gz) file containing the control read1 reads.
    • controlread2: The absolute path to the .FASTQ(.gz) file containing the control read2 reads.
    • description: A brief description of the sample

Pipeline Output

When running the full pipeline, the results of each step are outputted to the output_folder in a separate folder for each step. The output folders and their respective contents are as follows:

• output_folder/aligned: Contains an alignment .sam, alignment .bam, sorted bam, and .bai index file for each sample.
• output_folder/fastq: Merged .fastq.gz files for each sample.
• output_folder/identified: Contains tab-delimited .txt files for each sample containing the identified DSBs, control DSBs, filtered DSBs, and read quantification.
• output_folder/visualization: Contains a .svg vector image representing an alignment of all detected off-targets to the targetsite for each sample.

Testing the circleseq Package

In the spirit of Test-Driven Development, we have written tests for the pipeline to protect against regressions when making future updates. These can be used to ensure that the software is running with expected functionality.

NOTE: Due to differences in sorting between different versions of the bwa package, you must be using bwa 0.7.11 for these tests to work. We also recommend that you use samtools 1.3 when running these tests for consistency's sake.

Single-Step Regression Tests

For ongoing testing and development, we have created an abridged set of input data and expected output data for each step of the pipeline. This way, changes to the pipeline can be quickly tested for feature regression.

To run these tests, you must first install the nose testing Python package.

pip install nose

Then, from the circleseq root directory, simply run

nosetests

and the regression tests for for the pipeline will be run.

FAQ

None yet, we will keep this updated as needed.