Quality control by FastQC

• FastQC is a free and lightweight quality control app for checking the characteristics of sequencing data.
• It is used both for bulk and single-cell RNA-seq data.
• You can install it like any other apps, but you can run it from the command line too: fastqc MyExperiment.fastq

What can go (terribly) wrong with scRNA-seq data?

• In short: Infinite number of things
• Read complexity (same few genes are detected)
  • Messes up the sequencer’s cluster calling
    • Messes up base quality
• 

What can go (terribly) wrong with scRNA-seq data (#2)?

• Adapter sequences
  • Adapter dimers, primer dimers
  • Transpose sequence

Transposase sequence makes up 10% of the reads!

A kmer is a k-long sequence often found (e.g: the barcode)

Most common problem is decreasing Q over the read

Trimming Reads by Trim Galore!

• Trim Galore! is a wrapper for the reads trimming software cutadapt.
  • What is a wrapper?
  • What wrapper did you encounter already?
• trim_galore -h for help

Trimming

• You can trim on:
  • Quality
  • Custom defined / common adapter sequences to remove
• trim_galore --nextera MyExperiment.fastq Share/ERR522959_2.fastq
• Outputs a cleaned .fastq file
• Let’s rerun FastQC
• Once you are OK with QC, you are ready for mapping…
  • but…

Mapping is a computationally expensive step

So why not removing some more reads that would be useless anyways? >- Does your reads have the correct CBC and UMI? >- Pre-filtering reads for correct barcodes and UMI

Mapping (or alignment) is figuring out where each read is coming form

• Many tools have been developed
  • [If you have to choose: Just use the one that most people use, but look for up-comers]
• What do you need for mapping?
  • Your sequencing data (cleaned up)
  • Reference

Mapping Reference

• You can map to:
  • reference genome
  • reference transcriptome
• See an examples:

Reference genome

Is a .fasta file with 20-24 sequences: the chromosomes.

Reference transcriptome

When would you choose either of these?

• Speed
• Pseudogenes / multi-mappers
• Simplicity / convenience

What I mean about speed:

Organisation of the human genome

Mapping is an art on its own…

Why that? - It’s simple: >- Using R you could write it yourself > - compare base to base “A”=="A" > - your.base == base.in.reference > - in 2 gigantic for loops >- Comparing every base in your read vs every base in the reference is This is way to slow >- When you try to speed it up, it soon gets very complicated > - Creating a search index (lookup) for the reference is usually a key step >- There are a lot of other issues

Example ‘Splice aware’ alignment to catch reads that overlap exon junctions

• STAR is such an aligner

Practical steps

Two steps are required to perform STAR (or most other) alignments.

Step 1: Create a reference index

A search index for the reference is usually a key step

• a reference genome sequences (FASTA) and
• genome annotation file (GTF)
  • used to create a genome index
  • only for each genome/annotation combination

mkdir indices
mkdir indices/STAR
STAR --runThreadN 4 --runMode genomeGenerate --genomeDir indices/STAR --genomeFastaFiles Share/Reference.transcripts.fa

Step 2: Align your data

• Your reads (FASTQ) mapped to to the reference (FASTA) using the genome index (various files and extensions).

mkdir results
mkdir results/STAR

STAR --runThreadN 4 --genomeDir indices/STAR --readFilesIn Share/ERR522959_1.fastq Share/ERR522959_2.fastq --outFileNamePrefix results/STAR/

• After alignment you have a .sam file

Working with aligned data

• Conversion (depending on the file format)
• Counting
• Checking reads (if necessary)

Use samtools to convert .bam to .sam

Part of the HTSlib (High Throughput Sequencing Library )

SAMtools provide various utilities for manipulating alignments in the SAM format, including

• .bam / .sam conversion
• sorting,
• merging,
• indexing and
• generating alignments in a per-position format.

We wrote our own scripts to count reads per gene and cell, and finally create an expression matrix

→ We need a .sam file as input and the aligners typically generate a .bam file

Manually inspecting (mapped) files

• Sometimes necessary to check the raw data
  • You find an unexpected gene highly expressed
  • Check the raw reads
  • Maybe you find that reads mapping to that gene are essentially a poly-T stretch ATTTTTTTTTTTTTTTTTTTTN.
• You can best use linux command line tools
  • ‘less’, ‘more’, ‘nano’ and ‘vi’ can be used to inspect any text files from the command line.
• Use samtools to view

Commands to view files

You learned yesterday some commands:

head MyExperiment.fastq
nano MyExperiment.fastq

# After maooing
# You can use "samtools" to convert a .bam into a .sam
samtools view -h file.bam # creates a .sam

# Use pipe to count the number of lines in file.txt
# counts the number of lines in the samtools output
samtools view -h file.bam | wc -l

# Check the file
wc -l file.txt
samtools view -h file.bam | head | more

# Select a certain chromosome (if mapped against the genome)
samtools view -b in.bam chr1 > in_chr1.bam

# ...or per gene if mapped against the transcripome
samtools view -b in.bam Pou5f1__chr19 > in_chr1.bam

Creating the gene expression matrix

3 ways to count

• Read count (Simplest)
• UMI count (filtered for duplicates)
• Transcript count (using a statistic model for UMI sampling)

Sampling correction of UMI observations for transcript counting

The Number of UMI-s is not linearly correlated with the reads.

Why?

You are sequencing molecules from a large pool of molecules. This is a sampling process.

Imagine:

• Sampling marbles from a box
• marbles are of 256 colors
• In sampling with repacement, what is the chance that you see
  • 20 different UMI in 20 reads?
  • 200 different UMI in 200 reads?
• It is going to be less: you will start pulling the same color more than once…

Asking the reverse question to extrapolate the number of original mRNA molecules from the number of UMI!

• You could ask the reverse question: I see 20 different UMI in 25 reads, how many UMI were there in the box?

This is what we do in when we convert reads to transcript-counts.

Read more about the mapping process.

Our pipeline for mapping: MapAndGo2

Steps it automates

1. Unzipping everything
2. Concatenation of lanes
3. Extracting read and UMI info from R1.fastq and adding it into R2.fastq
4. Removal of cells with invalid CBC-s (cell barcodes) or UMI-s with N-s
5. Single end mapping
6. Conversion to count tables (usual UMI correction, etc)
7. Zipping up everything