DOC_ID : T11-0001
 

Doc_ID: A08-0001GRCh38en104Star275a
Editor: Mira
Reviewer: hsujc

Description

RSEM is a software package for estimating gene and isoform expression levels from RNA-Seq data. The RSEM package provides an user-friendly interface, supports threads for parallel computation of the EM algorithm, single-end and paired-end read data, quality scores, variable-length reads and RSPD estimation. In addition, it provides posterior mean and 95% credibility interval estimates for expression levels.

Build RSEM references using RefSeq, Ensembl, or GENCODE annotations
RefSeq and Ensembl are two frequently used annotations. For human and mouse, GENCODE annotaions are also available. Here, we show how to build RSEM references using Ensembl annotation. It is important to use every genome version with it’s compatible gtf file. 

Source

• URL :
  1. DNA Sequence :http://ftp.ensembl.org/pub/release-104/fasta/homo_sapiens/dna/=>Homo_sapiens.GRCh38.dna.primary_assembly.fa.gz
  2. GTF : http://ftp.ensembl.org/pub/release-104/gtf/homo_sapiens/=>Homo_sapiens.GRCh38.104.gtf.gz
• File size :
  1. Homo_sapiens.GRCh38.dna.primary_assembly.fa.gz 860.1MB (解壓縮後3.08G)
  2. Homo_sapiens.GRCh38.104.gtf.gz 48.5MB (解壓縮後1.30G)

Genome assemble version : GRCh38 Release 104

Detail information :

使用STAR version 2.7.5a及RSEM version 1.3.3 來製作index，請確認已完成GApp standard analysis environment的安裝

#Activate standard analysis environment

conda activate GApp

#移動到Ref資料夾

cd ~/GA_bundle/Ref/

#創建放置fasta及GTF的資料夾
mkdir -p Homo_sapiens/GRCh38en104Star2.7.5a/

#移動至資料夾

cd Homo_sapiens/GRCh38en104Star2.7.5a/

#下載fasta
wget ftp://ftp.ensembl.org/pub/release-104/fasta/homo_sapiens/dna/Homo_sapiens.GRCh38.dna.primary_assembly.fa.gz 

#解壓縮檔案
gunzip Homo_sapiens.GRCh38.dna.primary_assembly.fa.gz

#下載GTF
wget ftp://ftp.ensembl.org/pub/release-104/gtf/homo_sapiens/Homo_sapiens.GRCh38.104.gtf.gz

#解壓縮檔案
gunzip Homo_sapiens.GRCh38.104.gtf.gz

#用RSEM及STAR prepare index
rsem-prepare-reference \
–gtf ~/GA_bundle/Ref/Homo_sapiens/GRCh38en104Star2.7.5a/Homo_sapiens.GRCh38.104.gtf \
–star \
-p 20 \
~/GA_bundle/Ref/Homo_sapiens/GRCh38en104Star2.7.5a/Homo_sapiens.GRCh38.dna.primary_assembly.fa \
~/GA_bundle/Ref/Homo_sapiens/GRCh38en104Star2.7.5a/GRCh38.104.genome

使用gtfToGenePred及samtools version 1.10製作用來提供基因位置及rRNA位置的訊息的ref_flat file及 ribosomal intervals file

#利用gtfToGenePred將gtf轉為ref_flat檔

gtfToGenePred -genePredExt -geneNameAsName2 -ignoreGroupsWithoutExons Homo_sapiens.GRCh38.104.gtf /dev/stdout |awk ‘BEGIN { OFS=”\t”} {print $12, $1, $2, $3, $4, $5, $6, $7, $8, $9, $10}’ > Homo_sapiens.GRCh38.104.gtf.refflat

#由reference genome抽取genome大小資訊

#Step1
samtools faidx Homo_sapiens.GRCh38.dna.primary_assembly.fa

#Step2
cut -f1,2 Homo_sapiens.GRCh38.dna.primary_assembly.fa.fai > sizes.genome

#Step3
perl -lane ‘print “\@SQ\tSN:$F[0]\tLN:$F[1]\tAS:GRCh38″‘ sizes.genome |grep -v _ >> Homo_sapiens.GRCh38.104.gtf.rRNA.refflat

#合併由GTF抽出的rRNA資訊

grep ‘gene_biotype “rRNA”‘ Homo_sapiens.GRCh38.104.gtf |awk ‘$3 == “gene”‘ |cut -f1,4,5,7,9 |perl -lane ‘/gene_id “([^”]+)”/ or die “no gene_id on $.”;print join “\t”, (@F[0,1,2,3], $1)’ |sort -k1V -k2n -k3n >> Homo_sapiens.GRCh38.104.gtf.rRNA.refflat

Statistics

Summary

Gene counts (Primary assembly)

Gene counts (Alternative sequence)

Other

Index and modification

Index

Bundle files