Motivation

chimFinder is designed to identify reads from RNA and DNA sequencing experiments which align to two distinct genomes. This tool was developed as part of the work on identification of fusion transcripts resulting from HIV type 1 integrations in human CD4+ T cells.

Prerequisites

Along with chimFinder this repository contains several scripts designed to automate all steps of the pipeline described in the manuscript:

1. database cleanup (contamination.py)
2. read trimming and preprocessing
3. alignment to the genomes
4. full spliced landscape generation of the integrated genome
5. identification integration sites
6. final report

Several prerequisites are assumed to be installed and properly configured to be able to run the full pipeline

1. unix environment
2. bowtie
3. hisat
4. samtools
5. bowtie2 databases
6. hisat2 databases
7. trim galore (including fastq and cutadapt)
8. ncbi-toolkit

chimFinder uses as input two sam-formatted files. Users may choose to perform upstream analysis using other tools and pipelines which generate alignments in the appropriate sam format.

Installation

chimFinder is implemented in Python and assumes minimum requirements.

1. git clone
2. pip install -r requirements.txt (Optional) - wrapper.sh may be used to run the entire pipeline in an automated fashion. Please ensure all software is installed and properly configured on the machine
3. ./chimFinder.py -i1 <hiv.bowtie.sam> -i2 <hum.bowtie.sam> --spliced <hum.hisat.sam> -o <> -a <>

Parameters

-h, --help, help page

-i1r1 INPUT1R1, --input1r1 INPUT1R1 first alignment r1

-i1r2 INPUT1R2, --input1r2 INPUT1R2 first alignment r2

-i2r1 INPUT2R1, --input2r1 INPUT2R1 second alignment r1

-i2r2 INPUT2R2, --input2r2 INPUT2R2 second alignment r2

-o OUT, --out OUT
output file

-t THREADS, --threads THREADS the number of threads to use in the computation

--minCount MINCOUNT the minimum number of reads supporting an integration site.

--maxCountPenalty MAXCOUNTPENALTY the maximum penalty to give for minCount when hit.

--weightCount WEIGHTCOUNT Weight of the number of reads score in the cumulative score equation

--minEntropy MINENTROPY minimum alignment entropy score.

--weightEntropy WEIGHTENTROPY Weight of the entropy score in the cumulative score equation

--minQual MINQUAL minimum mean mapping quality of sequenced read. Everything below this threshold will be reported as multialignment for which no assumption can be made from the annotation

--weightQual WEIGHTQUAL Weight of the mapping score in the cumulative score equation

--minLen MINLEN the minimum number of nucleotides in alignment to keep a read.

--maxLenUnmapped MAXLENUNMAPPED minimum percent of the read to be aligned correctly in spanning read search

--weightLen WEIGHTLEN Weight of the alignment length score in the cumulative score equation

--steepSlopeAL STEEPSLOPEAL Weight of the alignment length score in the cumulative score equation

--maxAlLenPenalty MAXALLENPENALTY the maximum penalty to give for minLen when hit.

-s SCORE, --score SCORE the minimum overall score to keep.

--overlap OVERLAP overlap threshold

--gap GAP gap threshold

--close CLOSE distance between two integration sites to group together

-a ANNOTATION, --annotation ANNOTATION annotation for the human genome

-w, --writeReads write reads to fasta files

-p, --plot plot snapshots

-q, --quiet do not print to std out. report will still be saved

--splicedR1 SPLICEDR1 spliced end-to-end alignment r1. Reads will be subtracted from the main alignments and will not be reported in the final report of integrations sites

--splicedR2 SPLICEDR2 spliced end-to-end alignment r2. Reads will be subtracted from the main alignments and will not be reported in the final report of integrations sites

Output

chimFinder outputs the following three files per run:

1. .csv - standard ouput of integration sites grouped by position
2. .full.csv - same as .csv but contains additional information about the reads in each group, such as read name
3. .report - read statistics for each of the applied filters

Additional Scripts

sam2splice.py - used to extract relevant splice-sites. A dictionary of known HIV-1 (HXB2) donor and acceptor sites is hard-coded

-i, --input - spliced alignment in SAM format

-o, --output - output csv file with the results

-f , --filter - flag to filter out any splice junctions in the LTR region of HIV-1 as defined for the HXB2 genome.

--fasta - flag to output spliced reads to a separate file

runChimFinderOnAll.sh - used to run aligned samples in bulk through chimFinder, sam2splice.py and pileup

wrapper.sh - run the full pipeline from start to finish in bulk