seanchen607 / l1em Goto Github PK

You will need

1. git (https://git-scm.com/book/en/v2/Getting-Started-Installing-Git)
2. anaconda (https://docs.anaconda.com/anaconda/install/)

Download from github

git clone https://github.com/wmckerrow/L1EM

Create conda environment

cd L1EM
conda env create -f L1EM.yml

Before running L1EM, activate the environment:

source activate L1EM

When finished, deactivate the environment:

source deactivate L1EM

Alternatively you can install the following dependencies yourself:

• python version 2.7
• bwa (version 0.7.17 tested)
• samtools (version 1.9 tested)
• numpy (version 1.14.3 tested)
• scipy (version 1.1.0 tested)
• pysam (version 0.15.0 tested)
• bedtools (version 2.27.1 tested)

If necessary, you can specify the path for bwa and samtools in the run_L1EM.sh script. You must use samtools >=1.0. Early version of pysam will not work. I highly recommend that you use bwa 0.7.17. Earlier versions may differ in how they write the XA tag. This will lead to inaccurate results without throwing an error.

You will need the hg38 reference genome in fasta format, with bwa index. Downloaded from UCSC genome browser:

wget http://hgdownload.cse.ucsc.edu/goldenPath/hg38/bigZips/hg38.fa.gz
zcat hg38.fa.gz > hg38.fa
bwa index hg38.fa

Note: this will take some time.

Then you can build the L1EM reference using the provided shell script:

bash generate_L1EM_fasta_and_index.sh /fullpathto/hg38.fa

You will need a bam file with strand specific paired end read alignments to hg38. You can use any aligner, but make sure that all reads from the original fastq files are present trimming is okay, but filtering reads will potentially break the pipeline.

First move to an empty directory and then execute the shell script:

bash /fullpathto/run_L1EM.sh /fullpathto/alignments.bam /fullpathto/L1EM /fullpathto/hg38.fa

L1EM will write files with specific names, so do NOT run two instances of L1EM in the same directory.

At the end of the run_L1EM.sh script are a commented set of commands to delete all the intermediate files. If you wish to automatically delete intermediate files, you can delete these comments.

At completion, two tab delimited table will be written.

1. full_counts.txt
2. l1hs_transcript_counts.txt

The rows of both files are L1 loci.

For full_counts.txt each of the five transcript types: only, runon, passive (sense), passive (antisense), antisense are reported.

For l1hs_transcript_counts.txt only proper transcription from L1HS elements start at the 5' UTR is reported.

The results are also written as pickle files to facilitate further analysis in python. To generate a python dictionary with keys being the transcript names and values being the relative expression:

X_est = dict(zip(pickle.load(open('names_final.pkl')),pickle.load(open('X_final.pkl'))))

• URL for our methods paper will appear here when it is published
• More details can be found in manual.md