# Compare de novo reconstructed transcripts to reference annotations ### Download *Schizosaccharomyces pombe* genome and annotation ``` mkdir ~/LSLNGS2015/Trinity/GENOME_data cd ~/LSLNGS2015/Trinity/GENOME_data wget ftp://ftp.ensemblgenomes.org/pub/fungi/release-33/fasta/schizosaccharomyces_pombe/dna/Schizosaccharomyces_pombe.ASM294v2.dna.toplevel.fa.gz wget ftp://ftp.ensemblgenomes.org/pub/fungi/release-33/gff3/schizosaccharomyces_pombe/Schizosaccharomyces_pombe.ASM294v2.33.gff3.gz ``` ``` gunzip Schizosaccharomyces_pombe.ASM294v2.dna.toplevel.fa.gz gunzip Schizosaccharomyces_pombe.ASM294v2.33.gff3.gz samtools faidx Schizosaccharomyces_pombe.ASM294v2.dna.toplevel.fa ``` ## Builds the GMAP database ### Usage `gmap_build [options...] -d ` ### Execute ``` cd ~/LSLNGS2015/Trinity bsub -q 16G -o ./gmap_build.std -e ./gmap_build.err -J gmap_build \ "gmap_build -d gmap_spo -D . -k 13 Schizosaccharomyces_pombe.ASM294v2.dna.toplevel.fa" ``` ### Resource usage | ALPS Queue Name | CPU Time | Max Memory | Duration | | --------------- | ---------- | ---------- | ------------------- | | 16G | 78.21 sec. | - | 1 minute 41 seconds | ### Options `-d` Genome name. `-D` Destination directory. `-k` k-mer value for genomic index (allowed: 15 or less). ## Align Trinity transcripts to genome using GMAP ### Usage `gmap [OPTIONS...] , or cat | gmap [OPTIONS...]` ### Execute ``` cd ~/LSLNGS2015/Trinity bsub -q 16G -o ./gmap.std -e ./gmap.err -J gmap \ "gmap -t 6 -n 0 -D . -d gmap_spo trinity_reference/Trinity.fasta -f samse > trinity_gmap.sam" ``` ### Resource usage | ALPS Queue Name | CPU Time | Max Memory | Duration | | --------------- | ---------- | ---------- | --------- | | 16G | 11.90 sec. | - | 7 seconds | ### Options `-t` Number of worker threads. `-n` Maximum number of paths to show. If set to 0, GMAP reports single alignment plus chimeric alignments. `-D` Destination directory. `-d` Genome database. ### Convert SAM to sorted BAM and create index Convert SAM to BAM and sort by coordinates ``` bsub -q 16G -J sort \ "samtools sort -T trinity_gmap -o trinity_gmap.bam trinity_gmap.sam" ``` After BAM is generated, create index `samtools index trinity_gmap.bam` ## Builds the STAR database ``` cd ~/LSLNGS2015/Trinity mkdir star_spo bsub -q 16G -o ./star_build.std -e ./star_build.err -J star_build \ "STAR --runThreadN 6 --runMode genomeGenerate --genomeDir star_spo \ --genomeFastaFiles GENOME_data/Schizosaccharomyces_pombe.ASM294v2.dna.toplevel.fa" ``` ### Resource usage | ALPS Queue Name | CPU Time | Max Memory | Duration | | --------------- | ---------- | ---------- | ---------- | | 16G | 40.19 sec. | - | 29 seconds | ## Align RNA-Seq reads to genome using STAR ``` mkdir star_alignment bsub -q 16G -o ./star_map.std -e ./star_map.err -J star_map \ "STAR --genomeDir star_spo \ --readFilesIn RNASEQ_data/sp.left.fq.gz RNASEQ_data/sp.right.fq.gz \ --readFilesCommand zcat --outSAMtype BAM SortedByCoordinate --outFilterMultimapNmax 10 \ --outSAMunmapped None --twopassMode Basic \ --runThreadN 6 --outFileNamePrefix \"star_alignment/\"" ``` ### Resource usage | ALPS Queue Name | CPU Time | Max Memory | Duration | | --------------- | ----------- | ---------- | ------------------- | | 16G | 157.56 sec. | 4 GB | 1 minute 33 seconds | After BAM is generated, create index `samtools index star_alignment/Aligned.sortedByCoord.out.bam` ### List files Use `ls -la` to list the files in your Trinity folder ``` drwxrwxr-x 14 ycl6 ycl6 4096 Oct 27 12:52 . drwxrwxr-x 6 ycl6 ycl6 4096 Nov 1 13:44 .. drwxrwxr-x 2 ycl6 ycl6 4096 Oct 27 11:58 GENOME_data drwxrwxr-x 3 ycl6 ycl6 4096 Oct 27 10:55 gmap_spo drwxrwxr-x 2 ycl6 ycl6 4096 Oct 27 12:19 RNASEQ_data drwxrwxr-x 4 ycl6 ycl6 4096 Oct 27 12:36 star_alignment drwxrwxr-x 2 ycl6 ycl6 4096 Oct 27 12:32 star_spo -rw-rw-r-- 1 ycl6 ycl6 132734 Oct 27 12:25 trinity_gmap.bam -rw-rw-r-- 1 ycl6 ycl6 9872 Oct 27 12:27 trinity_gmap.bam.bai -rw-rw-r-- 1 ycl6 ycl6 454354 Oct 27 12:23 trinity_gmap.sam drwxrwxr-x 4 ycl6 ycl6 4096 Oct 27 12:41 trinity_reference -rw-rw-r-- 1 ycl6 ycl6 21181 Oct 27 12:22 trinity_reference.log ``` ## Visualize data using IGV The [Integrative Genomics Viewer (IGV)](http://software.broadinstitute.org/software/igv/) is a Java-based visualization tool for interactive exploration of large, integrated genomic datasets. ### Download data file to your computer Please use WinSCP or similar sFTP clients to download the following files from your off-site server such as **ALPS1** to local PC or laptop: ``` GENOME_data/Schizosaccharomyces_pombe.ASM294v2.dna.toplevel.fa GENOME_data/Schizosaccharomyces_pombe.ASM294v2.dna.toplevel.fa.fai GENOME_data/Schizosaccharomyces_pombe.ASM294v2.33.gff3 trinity_gmap.bam trinity_gmap.bam.bai star_alignment/Aligned.sortedByCoord.out.bam star_alignment/Aligned.sortedByCoord.out.bam.bai ``` ### Launch IGV via Java Web Start Go to the IGV downloads page: . When prompted, register or log in as requested. Click the **launch** icon to initiate the web start launch window. ![Install IGV](https://2175465529-files.gitbook.io/~/files/v0/b/gitbook-legacy-files/o/assets%2F-M44VuvT-GOxd2-xMygM%2F-M44Vv9Erqxz_QwrCvEL%2F-M44VzRNSKNOPPFwSCqR%2Figv1.png?generation=1586009223633005\&alt=media) ### After launching IGV 1. Go to **Genomes** -> **Load Genome from File**, and select *Schizosaccharomyces\_pombe.ASM294v2.dna.toplevel.fa*. Click Open. 2. Go to **File** -> **Load from File**, and select *Aligned.sortedByCoord.out.bam*, *trinity\_gmap.bam* and *Schizosaccharomyces\_pombe.ASM294v2.33.gff3*. Click Open. Change the chromosome range to `I:577,956-583,212`. In the window showing **Aligned.sortedByCoord.out.bam**, you can right click > Color alignments by > read strand, to change reads to Blue and Red colors for your paired-end reads. ![IGV in action](https://2175465529-files.gitbook.io/~/files/v0/b/gitbook-legacy-files/o/assets%2F-M44VuvT-GOxd2-xMygM%2F-M44Vv9Erqxz_QwrCvEL%2F-M44VzRPjRfwpR3QC_va%2Figv2.png?generation=1586009224630604\&alt=media)