SViper

This pipeline polishes deletions and isnertions called on long read data (ONT) using short exact reads for refinement.

Installation

For installation, simply clone the repo and use make to compile any utilities.

~$ git clone git@github.com:smehringer/SViper.git
~$ cd SViper
~$ make

Note: You need a compiler that supports c++14.

Dependencies

Currently, the polishing pipeline lacks a convinient long read pairwise alignment function. Therefore, the main program outputs a fasta file that needs to be mapped by a long read mapper of your choice.

Linux operating system
Code so far only tested for gcc 5.4.0
C++14 support
A long read mapper of your choice. Recommended: minimap2

Using SViper

You can look at all the input requirements by calling the sviper help page

~$ sviper -h

The usage of the whole polishing pipeline, needs a remapping step with a long read mapper of your choice, though. An example of using sviper is the following:

Call sviper

~$ sviper -s short-reads.bam -l long-reads.bam -r ref.fa -c variants.vcf -o example -g example.log

This will output a example.fa file, that contains all the polished sequences for recalling refined variants.

Map polished sequences to reference

~$ my-fav-mapper -input example.fa -r ref.fa

This will output a sam or bam file that serves as input for the third step.

Evaluate the mapping and create a polished VCF file IMPORTANT: The bam/sam file must be sorted by name in order for the evaluation to work correctly!

~$ samtools sort -n mapping-output.bam > mapping-output.sortedByName.bam
~$ evaluate_final_mapping mapping-output.sortedByName.bam variants.vcf

This will output a file called variants.vcf.polished.vcf containing variants with the same ID but refined break points or even failures if the variant was "polished away" (see Interpreting the output).

IMPORTANT

There are several requirements for using the polishing:

The vcf file must be a structural variant format (tags instead of sequences, e.g. <DEL>). ALso the INFO field must include the END tag, giving the end position of the variant, as well as the SVLEN tag in case of insertions.
The bam files must be indexed.
The reference sequence (FASTA) must be indexed.
BEOFRE you call evaluate_final_mapping you must sort the bam file by name!
(Obvisoulsy, the bam files should correspond to the same individual/sample mapped to the given reference.)

Input arguments:

-c, --candidate-vcf The candidate vcf file This file contains all the variants that will be polished in the run. If you want to accelarate polishing on a large vcf file, you might want to split the file into several small ones and call sviper each one seperately.
-s, --short-read-bam (BAM_FILE) The indexed bam file containing short used for polishing at variant sites.
-l, --long-read-bam (BAM_FILE) The indexed bam file containing long reads to be polished at variant sites.
-r, --reference (FA_FILE) The indexed (fai) reference file.
-o, --output-fa (FA_FILE) A filename for the output file. NOTE: The current output is a fasta file, that contains the polished sequences for each variant. Since the final realignment is not part of this tool yet,The user must map the fasta file with a mapper of his choice (e.g. minimap2) and then call evaluate_final_alignment.
-g, --log-file (TXT_FILE) A filename for the log file. Default: polishing.log.

-k, --flanking-region (INT) The flanking region in bp's around a breakpoint to be considered for polishing In range [50..1000]. Default: 400.

      ~~~~
                      start x             end y
      ------------------|------------------|----------------
                 !------------!      !------------!
                x-50        x+50    y-50         y+50
      ~~~~

Utilities

There are some utilities that come in handy when you want to look at specific variants and understand the substeps of polishing (or the preparation). You can make the utilities with make:

~$ make utilities

The executables can then be found in the utilities directory.

Some short explanation for each:

utilities_merge_split_alignments This little program will take a bam/sam file as first argument. It will then output a file called 'your-input-filename.merged.sam' which includes all reads but all supplementary alignments are now wither merged to it's primary or discarded. IMPORTANT: The input BAM file needs to be sorted by name! This is especially useful if you want to look at the sviper-output-bam file in the IGV browser (the merging is done automatically when evaluating).
utilities_get_supporting_reads This little program will take a bam/sam file as first argument and a vcf file as second (Note that it only reads the first variant because it is made for one variant only). It will then output each reads name and wether the read is supporting or not.
utilities_compare_vcf This little program will take a vcf file as first argument and a TRUTH vcf file as second. This script is not debugged enough so you might run into errors. If so please report them to me and I will try to fix them as soon as possible.

Contact

If you have any questions don't hesitate to contact me: svenja.mehringer[AT]gmail.com

hannespetur/SViper