SE-MEI

This repository contains a number of scripts and programs useful for finding mobile element insertion (MEI) sites from single-end datasets. In short, these programs align soft-clipped regions of alignments against repeat sequences and processes the results to find their locations.

Requirements

The pysam python module
bowtie1 or another short read aligner.
A local aligner, like STAR.
htslib 1.1, which is a submodule

Compilation

The C files in this repository can be compiled with the supplied Makefile by simply typeing make in the source directory.

General pipeline

Map with STAR or another local aligner against the genome.
Use extractSoftclipped on the resulting BAM file to create fastq files of the soft-clipped regions.
Download and filter the output from RepeatMasker from UCSC or another source. Filtering could be done as follows (piping to sed is only needed if you aligned to an Ensembl reference):
```
head -n 2 1/chr1.fa.out
for chrom in ls */
do
awk 'BEGIN{OFS="\t"}{if(NR>3) print $5,$6,$7,$11}' $chrom/*.out | sed 's/chr//g' >> rmask.bed
done
```
Extract the repeat sequences with bed2fasta.py using the just BED file from step 3. Note that this is a VERY slow program and should probably just be rewritten. One could alternatively just use bedtools getfasta -name, which is probably faster.
Align those results with bowtie1 or another short-read aligner to the repeat sequences.
Run compactRepeats on the resulting BAM file to create a quality controlled list in BED format of putative insertion sites.
Use bedtools cluster to group insertion sites into clusters. Ensure that the input is sorted (bedtools can be used for this).
Use filter.py to produce a BED file of only sites with at least two supporting alignments. A repeat type must be specified, since the output only contains a single repeat type.

A more detailed example

Below are the exact commands used to process a single example sample. The fastq file isn't provided as this is meant to simply be an illustrative example.

Align with STAR (note that multiple samples would be processed in this manner, so the index, which we assume has already been created, isn't unloaded until completion):

STAR --genomeDir indexes --genomeLoad LoadAndKeep --outSAMstrandField intronMotif --outFilterMultimapNmax 2 --outSAMattributes Standard --readFilesCommand zcat --readFilesIn foo.fq.gz --outFileNamePrefix foo --outStd SAM | samtools view -Sbo foo.bam -

Extract the soft-clipped sequences in fastq format:
```
extractSoftclipped foo.bam > foo.fq.gz
```

Download, filter and merge RepeatMasker sequences from UCSC (this example uses a mouse dataset and extract only LINE/L1 and SINE/Alu regions):

wget --quiet http://hgdownload.cse.ucsc.edu/goldenPath/mm10/bigZips/chromOut.tar.gz
tar xf chromOut.tar.gz
for d in `ls -d */`
do
    awk '{if(NR>3 && ($11=="LINE/L1" || $11=="SINE/Alu")) printf("%s\t%s\t%s\t%s\n",$5,$6,$7,$11)}' $d/*.out | sed -e 's/chr//g' >> rmask.bed #Convert UCSC to Ensembl chromosome names
done

Extract the genomic sequence of the repeat regions:

bed2fasta.py rmask.bed reference.fa > rmask.fa

Alternatively:

bedtools getfasta -name -fi reference.fa -bed rmask.bed -fo /dev/stdout | fold -w 60 > rmask.fa #This is faster than `bed2fasta.py`

Index and align to the repeat regions (with bowtie1 in this example):

bowtie-build rmask.fa rmask
bowtie -a --best --strata -p 6 rmask <(zcat foo.fq.gz) -S | samtools view -Sbo foo.bam - #You can ignore the "duplicated sequence" warnings

Perform a bit of QC and sort the sites:

compactRepeats foo.bam | bedtools sort > foo.bed #This could be merged with step 5 by specifying "-" as the input file.

Group reads into clusters (with multiple samples, concatenate the output from step 6 of each sample and then use bedtools sort before clustering):
```
bedtools cluster -d 50 foo.bed > foo.clustered.bed
```

Produce unique insertions of a single type

filter.py foo.clustered.bed "LINE/L1" foo.LINE.bed

Comparisons between groups or samples

A common goal is to find insertion sites unique to a given group of samples. This can be done as follows:

compareGroups Sample1.bed,Sample2.bed Sample3.bed,Sample4.bed All.bed > unique.bed

Note that compareGroups requires that the BED files are sorted in lexicographic order. Comparisons between samples can be done in a similar way, by specifying only a single file in one or both groups.

dpryan79 / SE-MEI