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Scripts for discovery and genotyping polymorphic Alu element insertions in human genomes

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AluMine

This GitHub repository stores various scripts required to discover and genotype polymorphic Alu element insertions. There are four different workflows: REF-plus discovery, REF-minus discovery, merging and filtering workflow and genotyping workflow. REF-plus and REF-minus workflows generate text file containing 32-mer pairs that can be subsequently used for genotyping using FastGT package. The scripts are written in PERL and bash.

Downloading to local server:

git clone https://github.com/bioinfo-ut/AluMine  

REF-plus discovery scripts

The key steps in REF-plus discovery pipeline are:

  • Search for all potential full-length Alu elements with the script find_ref_plus_candidates.pl. This script searches the reference genome for 10bp Alu element signatures (with 1 mismatch) and for Target Site Duplication sequences within 270-350 bp.
  • BLAST search that checks whether detected candidate elements are homologous to known Alu elements.
  • Search against chimpanzee genome using 25-mer lists. This step removes older elements that are likely to be fixed in both species.

To run the scripts yourself open pipeline_ref_plus.sh in text editor and define paths to FASTA files of the reference genome and chimpanzee genome.

cd ~/AluMine/discovery_REF-plus
bash pipeline_ref_plus.sh  

REF-minus discovery scripts

The key elements in REF-minus discovery pipeline are: find_ref_minus_candidates_bam.pl, gtester, and ref_minus_post_gtester.pl.

  • Scripts find_ref_minus_candidates_bam.pl or find_ref_minus_candidates_fastq.pl search for 10bp Alu signature sequences from BAM or FASTQ files and write out all potential signatures together with 25bp flanking sequence.
  • Then 'gtester' is used to localize the 25bp flanking sequences in the reference genome.
  • ref_minus_post_gtester.pl removes fixed Alu elements (present in the human reference genome) and prints out pair of 32-mers for those that are not present in reference genome.

Before running the REF-minus discovery pipeline, open file pipeline_ref_minus.sh in text editor and define paths to sample files and path to human chromosome files.

cd ~/AluMine/discovery_REF-minus
bash pipeline_ref_minus.sh

pipeline_ref_minus.sh supports input in BAM, single FASTQ, multiple FASTQ and gnuzipped FASTQ formats.

Merging and filtering the k-mer databases

The following steps are required for additional filtering using genotype data from real individuals

  • Alu-element candidates with identical k-mers were removed.
  • Alu-element candidates that are located within 25bp of each other were removed.
  • REF-minus and REF-plus k-mers need to be merged with 30M SNV k-mers. SNVs help to build more accurate model for genotype calling.

Remaining Alu-elements were genotyped on 2200 individuals and additional filtering steps were used:

  • Alu elements with >10% calls with unexpected ploidy were removed.
  • Alu elements with Hardy-Weinberg Equilibrium P-value<1.5E-6 were removed.

These steps are performed separately, their description can be seen in file filter_gt.pl.

cd ~/AluMine/discovery_merging_and_filtering
more pipeline_merging_and_filtering.sh

Genotyping

It is possible to skip the discovery phase and use our database of known Alu insertion polymorphisms (32,786 candidate polymorphisms).
The k-mer database for genotyping (ALU_v1.kmer.db) is available at FastGT webpage.

cd ~/AluMine/genotyping
bash pipeline_genotyping.sh

Citing

Please cite: Puurand T, Kukuškina V, Pajuste F-D, Remm M. (2019). AluMine: alignment-free method for the discovery of polymorphic Alu element insertions. Mobile DNA 10:31. [https://doi.org/10.1186/s13100-019-0174-3](doi: https://doi.org/10.1186/s13100-019-0174-3).

Additional data

Additional material can be downloaded from our webpage at http://bioinfo.ut.ee/?page_id=167&lang=en. Pre-compiled human 25-mer index for REF-minus discovery (57GB) and pre-compiled chimp 32-mer list for REF-plus discovery (27GB) can be downloaded from http://bioinfo.ut.ee/AluMine/.

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