Genome annotations

Genome annotations are in GFF format GFF format contains genes and other genomic features. Allows “track” info for visualization http:\/\/useast.ensembl.org\/info\/website\/upload\/gff.html

Visualizing and annotating Genomes

Gbrowse is a comprehensive database + interactive web application for manipulating and displaying annotations on genomes.

Genomics pipeline

Outlined below are the steps taken to create a raw vcf file from paired end raw FASTQ files. This was done for each sequenced accession so a HTCondor DAG Workflow was written to streamline the processing of those ~200 accessions. While some cpu and memory parameters have been included within the example steps below those parameters varied from sample to sample and the workflow has been honed to accomodate that variation. This pipeline is subject to modification based on software updates and changes to software best practices.

Software versions:

Preparing reference genome

Download Sorghum bicolor v3.1 from Phytozome

Generate:

BWA index:

bwa index –a bwtsw Sbicolor_313_v3.0.fa

fasta file index:

samtools faidx Sbicolor_313_v3.0.fa

sequence dictionary:

java –jar picard.jar CreateSequenceDictionary R=Sbicolor_313_v3.0.fa O=Sbicolor_313_v3.0.dict

Quality trimming and filtering of paired end reads

java –jar Trimmomatic-0.35/trimmomatic-0.35.jar PE -phred33 -trimlog trimlogPE.txt SampleA_R1.fastq.gz SampleA_R2.fastq.gz SampleA_R1.PE.fastq.gz SampleA_R1.SE.fastq.gz SampleA_R2.PE.fastq.gz SampleA_R2.SE.fastq.gz ILLUMINACLIP:adapters.fa:2:30:10 SLIDINGWINDOW:5:20 LEADING:20 TRAILING:20 MINLEN:60 2> trim.out

Aligning reads to the reference

bwa mem –M –R “@RG\tIDSAMPLEA_RG1\tPL:illumina\tPU:FLOWCELL_BARCODE.LANE.SAMPLE_BARCODE_RG_UNIT\tLB:libraryprep-lib1\tSM:SAMPLEA” Sbicolor_313_v3.0.fa SampleA_R1.PE.fastq.gz SampleA_R2.PE.fastq.gz > SAMPLEA.bwa.sam

Convert and Sort bam

Samtools view –bS SAMPLEA.bwa.sam | samtools sort - SAMPLEA.bwa.sorted

Mark Duplicates

java –Xmx8g –jar picard.jar MarkDuplicates MAX_FILE_HANDLES_FOR_READ_ENDS_MAP=1000 REMOVE_DUPLICATES=true INPUT=SAMPLEA.bwa.sorted.bam OUTPUT=SAMPLEA.dedup.bam METRICS_FILES=SAMPLEA.dedup.metrics

Index bam files

samtools index SAMPLEA.dedup.bam

Find intervals to analyze

java –Xmx8g –jar GenomeAnalysisTK.jar –T RealignerTargetCreator –R Sbicolor_313_v3.0.fa –I SAMPLEA.dedup.bam –o SAMPLEA.realignment.intervals

Realign

java –Xmx8g –jar GenomeAnalysisTK.jar –T IndelRealigner –R Sbicolor_313_v3.0.fa –I SAMPLEA.dedup.bam –targetIntervals SAMPLEA.realignment.intervals –o SAMPLEA.dedup.realigned.bam

Variant Calling with GATK HaplotypeCaller

java –Xmx8g –jar GenomeAnalysisTK.jar –T HaplotypeCaller –R Sbicolor_313_v3.0.fa –I SAMPLEA.dedup.realigned.bam --emitRefConfidence GVCF --pcr_indel_model NONE -o SAMPLEA.output.raw.snps.indels.g.vcf

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