--- title: "Genetic Variation Annotation Pipeline" canonical: "https://help.biobam.com/space/OBD/3292790785/Genetic%20Variation%20Annotation%20Pipeline" format: markdown --- # Introduction Genetic Variation Analysis of *Glycine Max.* ### Dataset Description This dataset contains 301 Canadian soybean lines that were subjected to GBS analysis (with *Ape*KI digestion). - Organism: *[Glycine Max](https://www.ncbi.nlm.nih.gov/data-hub/taxonomy/3847/)**.* - Instrument: Illumina HiSeq 2000. - Layout: Paired-end. ### Publication [Torkamaneh, Davoud, Jérôme Laroche, and François Belzile. "Genome-wide SNP calling from genotyping by sequencing (GBS) data: a comparison of seven pipelines and two sequencing technologies." ](https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0131533#sec002)*[PloS one](https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0131533#sec002)*[ 11.8 (2016): e0161333.](https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0131533#sec002)
Abstract In this work we describe a comprehensive comparison of seven GBS bioinformatics pipelines developed to process raw GBS sequence data into SNP genotypes. We compared five pipelines requiring a reference genome (TASSEL-GBS v1& v2, Stacks, IGST, and Fast-GBS) and two *de novo* pipelines that do not require a reference genome (UNEAK and Stacks). Using Illumina sequence data from a set of 24 re-sequenced soybean lines, we performed SNP calling with these pipelines and compared the GBS SNP calls with the re-sequencing data to assess their accuracy. The number of SNPs called without a reference genome was lower (13k to 24k) than with a reference genome (25k to 54k SNPs) while accuracy was high (92.3 to 98.7%) for all but one pipeline (TASSEL-GBSv1, 76.1%). Among pipelines offering a high accuracy (>95%), Fast-GBS called the greatest number of polymorphisms (close to 35,000 SNPs + Indels) and yielded the highest accuracy (98.7%).
> ℹ️ In order to accelerate the process, a subset of 23 samples was used. ### Original Data - Illumina ENA Run: [PRJNA287266](https://www.ncbi.nlm.nih.gov/bioproject/PRJNA287266). - NCBI Genome: [Glycine max (soybean)](https://www.ncbi.nlm.nih.gov/genome/?term=glycine+max). # Bioinformatic Analysis ## 1 - DNA-Seq Alignment ### Application [DNA-Seq Alignment](https://biobam.atlassian.net/wiki/spaces/OBD/pages/2488008844) (BWA). ### Input - [Illumina sequencing data](https://drive.google.com/drive/folders/1lg-0aP6u9N-OdGzGdHb-H8a9onYbhl8V?usp=share_link) in FASTQ format. - [NCBI genome](https://drive.google.com/file/d/1AHyphdtw5-o4uTYfsZwWOjWmo_tGzzmj/view?usp=share_link) in FASTA format. ### Parameters ### Execution Time Around 30 minutes. ### Output - [BAM files containing short read alignments.](https://drive.google.com/drive/folders/1fOAS-eR7yLJQ04BwhmaBgP_c6zGKci-s?usp=share_link) - [alignments_per_category_bwa.box: ](https://drive.google.com/file/d/1ucHq9Q3iTF8sIxr1KdYUyRsNxZ08QFRM/view?usp=sharing)Stacked bar plot about the DNA-Seq Alignments results. - [relative_alignments_per_category.box:](https://drive.google.com/file/d/1i5KNkNw5j_sZzM-DmOdfIMmjsd0jhlMA/view?usp=sharing) Stacked bar plot about the DNA-Seq Alignment results (percentages). - [report_bwa.box:](https://drive.google.com/file/d/1DrsEHATeLFVN20nRmdCW1hoyqvP0cmnL/view?usp=sharing) Report about the DNA-Seq Alignment results. ## 2- Variant Calling ### Application [Variant Calling by BCFtools](https://biobam.atlassian.net/wiki/spaces/OBD/pages/1117356121). ### Input - [NCBI genome](https://drive.google.com/file/d/1AHyphdtw5-o4uTYfsZwWOjWmo_tGzzmj/view?usp=share_link) in FASTA format. - [DNA-Seq Alignments](https://drive.google.com/drive/folders/1fOAS-eR7yLJQ04BwhmaBgP_c6zGKci-s?usp=share_link) in BAM format (from the [1 - DNA-Seq Alignment](https://biobam.atlassian.net/wiki/spaces/OBD/pages/2434465807/Long+Read+Prokaryotic+Analysis#2---DNA-Seq-Alignment) step). ### Parameters ### Execution Time 16 minutes. ### Output - [bcftools.vcf.gz:](https://drive.google.com/file/d/1TxY86AjdIwRTnS1LRClC_CQy1FZokeOe/view?usp=share_link) VCF File. - [variant_calling_report.box:](https://drive.google.com/file/d/1cnkebhocPg_TQgGT-648LC8WGX3dx8mE/view?usp=share_link) Summary report about variants found in alignments. - [raw_read_depth.box:](https://drive.google.com/file/d/1Z-tWdGpt18MwCOs8bE7FA03g806R0Y-s/view?usp=share_link) Distribution of raw depths. - [proportion_quality_depth.box:](https://drive.google.com/file/d/1I15gzJn91x8ALt9H2eRp1dlUGKvVI1Jr/view?usp=share_link) Distribution of the proportion quality/raw depth. - [average_mapping_quality.box:](https://drive.google.com/file/d/1fufD3IlVm6uOG3KPqp3Sy2SnZuj4_nCC/view?usp=share_link) Distribution of the MQ field. ## 3- Variant Filtering ### Application Variant Filtering. ### Input - [VCF file](https://drive.google.com/file/d/1TxY86AjdIwRTnS1LRClC_CQy1FZokeOe/view?usp=share_link) (from the [2- Variant Calling step](https://biobam.atlassian.net/wiki/spaces/OBD/pages/2434465807/Long+Read+Prokaryotic+Analysis#3--Polishing)). ### Parameters - Proportion ‘Quality/Counts’: 2 - Raw Read Depth: 2 - Phred Quality: 20 - Average Mapping Quality: 59 - Remove Multiple Alleles: True - Missing Genotypes Per Variant: 0 - Genotype Depth Threshold: 1 - Genotype Quality Threshold: 0 - Minimum Allele Frequency Threshold: 0.05 - Check Reads in Both Strands: False - Check if Reads are Balanced: False ### Execution Time 2 seconds. ### Output - [filtered.vcf.gz:](https://drive.google.com/file/d/1QKbCm-noJi-4RJ3nbb47r3WqBEp-xRP4/view?usp=drive_link) Filtered VCF File. - [variant_filtering_report.box:](https://drive.google.com/file/d/1PxtjxrCnDEIvtW1SeQ7oDRxjIUUH2zYJ/view?usp=drive_link) Summary report about the filtering step. - [raw_read_depth.box:](https://drive.google.com/file/d/1zNtFswyVGj3_PZUjXmm4MRF52mwvsqV1/view?usp=share_link) Distribution of raw depths in the variant that passed the filter. - [phred_quality.box:](https://drive.google.com/file/d/1joZXImJdJftwdq837WslQOmNuYUoEXKY/view?usp=sharing) Distribution of the QUAL field. - [proportion_quality_depth.box:](https://drive.google.com/file/d/1zHVrV_VljoRvv1pzEZcOeoHRUAT-gN_K/view?usp=sharing) Distribution of the proportion quality/raw depth. - [average_mapping_quality.box:](https://drive.google.com/file/d/1wO-_DymihFkiw9v4vSApQBtk23KatX0b/view?usp=sharing) Distribution of the MQ field. - [maf_histogram.box:](https://drive.google.com/file/d/1RC23Xu6Me8zHm1Dc1KzYzQQLNl3WlzbA/view?usp=sharing) Distribution of MAF Values. ## 4- Variant Annotation ### Application: Variant Annotation using VEP. ### Input: - [Filtered VCF](https://drive.google.com/file/d/1flrQt885Bhh-uT3oFd7lYSU5BzdEihe-/view?usp=share_link) (from the [3- Variant Filtering step](https://biobam.atlassian.net/wiki/spaces/OBD/pages/2434465807/Long+Read+Prokaryotic+Analysis#4--Gene-Finding)). - [Genome](https://drive.google.com/file/d/1AHyphdtw5-o4uTYfsZwWOjWmo_tGzzmj/view?usp=share_link) - [Annotation File](https://drive.google.com/file/d/1HAhWSrhwjOsCqIJwQ93NJSLSezgdS7xT/view?usp=share_link) ### Execution Time 10 minutes. ### Output - [annotation.box:](https://drive.google.com/file/d/1UpNOW3fPkAWR6lO_pPGph24ASlzrOJgz/view?usp=sharing) Table with information of each found variant. - [report.box: ](https://drive.google.com/file/d/13qBuiNdfku8zwq_zakUr0UPvghJUncAn/view?usp=sharing)Summary report with information of the type of variants, their consequences and some population genetics information. # Workflow ![image](media://a088b659-33af-456f-8ee2-82b9aa5671c7)