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Reference Genomes | Griffith Lab

## RNA-seq Bioinformatics

Introduction to bioinformatics for RNA sequence analysis

# Reference Genomes

### Obtain a reference genome from Ensembl, iGenomes, NCBI or UCSC.

In this example analysis we will use the human GRCh38 version of the genome from Ensembl. Furthermore, we are actually going to perform the analysis using only a single chromosome (chr22) and the ERCC spike-in to make it run faster…

Create the necessary working directory

cd $RNA_HOME echo$RNA_REFS_DIR
mkdir -p $RNA_REFS_DIR The complete data from which these files were obtained can be found at: ftp://ftp.ensembl.org/pub/release-86/fasta/homo_sapiens/dna/. You could use wget to download the Homo_sapiens.GRCh38.dna_sm.primary_assembly.fa.gz file, then unzip/untar. This has been done for you and that data placed on your AWS instance. It contains chr22 and ERCC transcript fasta files in both a single combined file and individual files. Copy the file to the rnaseq working directory cd$RNA_REFS_DIR
wget http://genomedata.org/rnaseq-tutorial/fasta/GRCh38/chr22_with_ERCC92.fa
ls

View the first 10 lines of this file. Why does it look like this?

How many lines and characters are in this file? How long is this chromosome (in bases and Mbp)?

wc chr22_with_ERCC92.fa

View 10 lines from approximately the middle of this file. What is the significance of the upper and lower case characters?

head -n 425000 chr22_with_ERCC92.fa | tail

What is the count of each base in the entire reference genome file (skipping the header lines for each sequence)?

cat chr22_with_ERCC92.fa | grep -v ">" | perl -ne 'chomp $_;$bases{$_}++ for split //; if (eof){print "$_ $bases{$_}\n" for sort keys %bases}'

Note: Instead of the above, you might consider getting reference genomes and associated annotations from UCSC. e.g., UCSC GRCh38 download.

Wherever you get them from, remember that the names of your reference sequences (chromosomes) must those matched in your annotation gtf files (described in the next section).

Assignment: Use a commandline scripting approach of your choice to further examine our chr22 reference genome file and answer the following questions.

Hint: Each question can be tackled using approaches similar to those above, using the file ‘chr22_with_ERCC92.fa’ as a starting point. Hint: To make things simpler, first produce a file with only the chr22 sequence. Questions

How many bases on chromosome 22 correspond to repetitive elements? What is the percentage of the whole length? How many occurences of the EcoRI restriction site are present in the chromosome 22 sequence?

Solution: When you are ready you can check your approach against the Solutions.

Introduction to Inputs | Griffith Lab

## RNA-seq Bioinformatics

Introduction to bioinformatics for RNA sequence analysis

# Introduction to Inputs

### Module 1 - Key concepts

• Review central dogma, RNA sequencing, RNAseq study design, library construction strategies, biological vs technical replicates, alignment strategies, etc.

### Module 1 - Learning objectives

• Introduction to the theory and practice of RNA sequencing (RNA-seq) analysis
• Rationale for sequencing RNA
• Challenges specific to RNA-seq
• General goals and themes of RNA-seq analysis work flows
• Common technical questions related to RNA-seq analysis
• Getting help outside of this course
• Introduction to the RNA-seq hands on tutorial

In...

• ## Introduction to Inputs

### Module 1 - Key concepts

• Review central dogma, RNA sequencing, RNAseq study design, library construction strategies, biological vs...