MICR5831 Module 2 Tutorial 2

What is sequencing depth

number of times that a given nucleotide in the genome has been read in an experiment

The total number of sequence reads generated for a sample during 16S rRNA sequencing or shotgun sequencing

standardised number of reads per sample used during downstream analyses

What happens when sampling depth is uneven

artificial inflation of diversity

Rare taxa more likely to be found with higher sampling depth

Full range of species present in the sample is rarely saturated

Why do we normalise the feature table

to eliminate bias due to differences in the sampling sequencing depth

Biases do not reflect true differences in the underlying biology but exist due to variations in sample collection, library preparation, and/or sequencing

Effective normalisation enables accurate comparisons of statistics from different measurements

What happens when there is low sampling depth

Loss of rare taxa

List all normalisation methods

Centre log ratio (CLR)

Total sum Scaling (TSS)

Rarefaction

What is Centre log ratio 

Normalisation + transformation
Normalisation = removes per-sample technical effects (e.g. sampling depth)
Transformation = makes skewed data nicer so the model fits are valid

Applies a centred log ratio transformation

Advantage of CLR

Suitable for the study of correlation coefficients and subsequent multivariate data analyses

Log-ratios are often preferable as they are resilient to matrix effects.

What is TSS

The most common normalisation technique

Transforms the feature table into proportions (relative abundance)

Divides feature read counts (the number of reads that cluster within the same ASV) by the total number of reads in each sample

What is rarefaction

randomly subsampling each sample to the lowest read depth of any sample.Or up until the same pre-defined number to ensure all samples have the same sampling depth

Advantage of rarefaction

low depth samples contain a higher proportion of contaminants (rRNA not from the intended sample) and they will be removed if they don’t meet the sampling depth defined

Disadvantage of rarefaction

this technique can throw out a lot of data, so it is not always appropriate

What is Alpha diversity

A measure of how diverse a single sample is, usually taking into account the number of different species observed.

What are the components of Alpha diversity

Richness and Evenness

What is Richness

estimates the number of different species present in a sample

It only takes into account the absence and presence of taxa

What is used to measure species richness

Chao1

Observed Feature

Faith PD

What is Chao1

Chao1 is an indicator of species richness

Estimates the total number of species in a sample

What 3 factors does Chao1 take into account

The number of species

The number of singleton taxa

The number of doubleton taxa.

What is Observed features

Indicator of species richness

The number of taxa (ASVs) observed

What is the difference between Chao1 and Observed feature

Chao1 considers not only observed ASVs, gives more weight to the lowabundance taxa, The singletons and doubletons are used to estimate the number of missing taxa

While

Observed features only considers observed ASVs.

What is Faith PD

Phylogenetic diversity (PD) is a measure of diversity, based on phylogeny

defined as the sum of the branch lengths of a phylogenetic tree connecting all species covered by a sample

Most widely used phylogenetic metric

Measures species richness

What is Evenness

a measure of relative abundance of different species that make up the richness.

What can be used to measure Evenness

Pielou’s evenness

Shannon Diversity Index

Simpson Index

What is Pielou’s Evenness

Provides information about the equity in species abundance in each sample,

Evenness is high if all species have similar distribution

A low J (evenness score) indicates that 1 or few species dominate the community

What is Shannon Diversity

Provides information on both species richness and evenness

Takes into account the relative abundance of each species

Considers the rarity and commonness of species in a community

Assumes all species are represented in a sample and that they are randomly sampled

Values of H are usually between 1.5 - 3.5 (the units are bits of information)

A value of H = 0 indicates a community that only has one species
doesnt Exceed 5 generally

What is Simpson Index

Provides information on both species richness and evenness

A dominance index – it gives more weight to common or dominant species.

• A few rare species with only a few representatives will not affect the diversity