# Glossary & FAQ¶

## Glossary of terms used in inStrain¶

Note

This glossary is meant to give a conceptual overview of the terms used in inStrain. See Expected output for explanations of specific output data.

ANI
Average nucleotide identity. The average nucleotide distance between two genomes or .fasta files. If two genomes have a difference every 100 base-pairs, the ANI would be 99%
conANI
Consensus ANI - average nucleotide identity values calculated based on consensus sequences. This is commonly reported as “ANI” in other programs. Each position on the genome is represented by the most common allele (also referred to as the consensus allele), and minor alleles are ignored.
popANI

Population ANI - a new term to describe a unique type of ANI calculation performed by inStrain that considers both major and minor alleles. If two populations share any alleles at a loci, including minor alleles, it does not count as a difference when calculating popANI. It’s easiest to describe with an example: consider a genomic position where the reference sequence is ‘A’ and 100 reads are mapped to the position. Of the 100 mapped reads, 60 have a ‘C’ and 40 have an ‘A’ at this position. In this example the reads share a minor allele with the reference genome at the position, but the consensus allele (most common allele) is different. Thus, this position would count as a difference in conANI calculations (because the consensus alleles are different) and would not count as a difference in popANI calculations (because the reference sequence is present as an allele in the reads). See Important concepts for examples.

Representative genomes (RGs) are genomes that are used to represent some taxa. For example you could have a series of representative genomes to represent each clade of E. coli (one genome for each clade), or you could have one representative genome for the entire species of E. coli (in that case it would be a Species Representative Genome (SRG)). The base unit of inStrain-based analysis is the representative genome, and they are usually generated using the program dRep

Species representative genome
A Species Representative Genome (SRG) is a representative genome that is used to represent an entire single microbial species.
Genome database
A collection of representative genomes that are mapped to simultaneously (competitive mapping).
nucleotide diversity
A measurement of genetic diversity in a population (microdiversity). We measure nucleotide diversity using the method from Nei and Li 1979 (often referred to as ‘pi’ π in the population genetics world). InStrain calculates nucleotide diversity at every position along the genome, based on all reads, and averages values across genes / genomes. This metric is influenced by sequencing error, but within study error rates should be consistent and this effect is often minor compared to the extent of biological variation observed within samples. This metric is nice because it is not affected by coverage. The formula for calculating nucleotide diversity is the sum of the frequency of each base squared: 1 - [(frequency of A)^2 + (frequency of C)^2 + (frequency of G)^2 + (frequency of T)^2 ].
microdiversity
We use the term microdiversity to refer to intraspecific genetic variation, i.e. the genetic variation between cells within a microbial species.
clonality
The opposite of nucleotide diversity (1 - nucleotide diversity). A deprecated term used in older versions of the program.
SNV
Single nucleotide variant. A single nucleotide change that is present in a faction of a population. Can also be described as a genomic loci with multiple alleles present. We identify and call SNVs using a simple model to distinguish them from errors, and more importantly in our experience, careful read mapping and filtering of paired reads to be assured that the variants (and the reads that contain them) are truly from the species being profiled, and not from another species in the metagenome (we call it ‘mismapping’ when this happens). Note that a SNV refers to genetic variation within a read set.
SNS
Single nucleotide substitution. A single nucleotide change that has a fixed difference between two populations. If the reference genome has a ‘A’ at some position, but all of the reads have a ‘C’ at that position, that would be a SNS (if half of the reads have an ‘A’ and half of the reads have a ‘C’, that would be an SNV).
divergent site
A position in the genome where either an SNV or SNS is present.
SNP
Single Nucleotide Polymorphism. In our experience this term means different things to different people, so we have tried to avoid using it entirely (instead referring to SNSs, SNVs, and divert sites).
A measure of how likely two divergent sites are to be inherited together. If two alleles are present on the same read, they are said to be “linked”, meaning that they are found together on the same genome. Loci are said to be in “linkage disequilibrium” when the frequency of association of their different alleles is higher or lower than what would be expected if the loci were independent and associated randomly. In the context of microbial population genetics, linkage decay is often used as a way to detect recombination among members of a microbial population. InStrain uses the metrics r2 (r squared) and D’ (D prime) to measure linkage.
coverage
A measure of sequencing depth. We calculate coverage as the average number of reads mapping to a region. If half the bases in a scaffold have 5 reads on them, and the other half have 10 reads, the coverage of the scaffold will be 7.5
A measure of how much of a region is covered by sequencing reads. Breadth is an important concept that is distinct from sequencing coverage, and gives you an approximation of how well the reference sequence you’re using is represented by the reads. Calculated as the percentage of bases in a region that are covered by at least a single read. A breadth of 1 means that all bases in a region have at least one read covering them
relative abundance
The percentage of total reads that map a particular entity. If a metagenome has 1,000,000 reads and 1,000 reads to a particular genome, that genome is at 0.1% relative abundance
contig
A contiguous sequence of DNA. Usually used as a reference sequence for mapping reads against. The terms contig and scaffold are used interchangeably by inStrain.
scaffold
A sequence of DNA that may have a string of “N”s in it representing a gap of unknown length. The terms contig and scaffold are used interchangeably by inStrain.
iRep
A measure of how fast a population was replicating at the time of DNA extraction. Based on comparing the sequencing coverage at the origin vs. terminus of replication, as described in Brown et. al., Nature Biotechnology 2016
mutation type
Describes the impact of a nucleotide mutation on the amino acid sequence of the resulting protein. N = non-synonymous mutation (the encoded amino-acid changes due to the mutation). S = synonymous mutation (the encoded amino-acid does not change due to the mutation; should happen ~1/6 of the time by random chance due to codon redundancy). I = intergenic mutation. M = multi-allelic SNV with more than one change (rare).
dN/dS
A measure of whether the set of mutations in a gene are biased towards synonymous (S) or non-synonymous (N) mutations. dN/dS is calculated based on mutations relative to the reference genome. dN/dS > 1 means the bias is towards N mutations, indicating the gene is under active selection to mutate. dN/dS < 1 means the bias is towards S mutations, indicated the gene is under stabilizing selection to not mutate. dN/dS = 1 means that N and S mutations are at the rate expected by mutating positions randomly, potentially indicating the gene is non-functional.
pN/pS
Very similar to dN/dS, but calculated at positions with at least two alleles present rather than in relation to the reference genome.
fasta file
A file containing a DNA sequence. Details on this file format can be found on wikipedia
bam file
A file containing metagenomic reads mapped to a DNA sequence. Very similar to a .sam file. Details can be found online
scaffold-to-bin file
A .text file with two columns separated by tabs, where the first column is the name of a scaffold and the second column is the name of the bin / genome the scaffold belongs to. Can be created using the script parse_stb.py that comes with the program dRep See Expected output for more info
genes file
A file containing the nucleotide sequences of all genes to profile, as called by the program Prodigal. See Expected output for more info
A read that is erroneously mapped to a genome. InStrain profiles a population by looking at the reads mapped to a genome. These reads are short, and sometimes reads that originated from one microbial population map to the representative genome of another (for example if they share homology). There are several techniques that can be used to reduce mismapping to the lowest extent possible.
A read that maps equally well to multiple different locations in the .fasta file. Most mapping software will randomly select one position to place multi-mapped reads. There are several techniques that can be used to reduce multi-mapped reads to the lowest extent possible, including increasing the minimum MAPQ cutoff to >2 (which will eliminate them entirely).
inStrain profile
An inStrain profile (aka IS_profile, IS, ISP) is created by running the inStrain profile command. It contains all of the program’s internal workings, cached data, and is where the output is stored. Additional commands can then be run on an IS_profile, for example to analyze genes, compare profiles, etc., and there is lots of nice cached data stored in it that can be accessed using python.
null model
The null model describes the probability that the number of true reads that support a variant base could be due to random mutation error, assuming Q30 score. The default false discovery rate with the null model is 1e-6 (one in a million).
mm
The maximum number of mismatches a read-pair can have to be considered in the metric being considered. Behind the scenes, inStrain actually calculates pretty much all metrics for every read pair mismatch level. That is, only including read pairs with 0 mismatches to the reference sequences, only including read pairs with >= 1 mis-match to the reference sequences, all the way up to the number of mismatches associated with the “PID” parameter. Most of the time when it then generates user-facing output, it uses the highest mm possible and deletes the column label. If you’d like access to information on the mm-level, see the section titled “Dealing with mm”
mapQ score
MapQ scores are a measure of how well a read aligns to a particular position. They are assigned to each read mapped by bowtie2, but the details of how they are generated are incredibly confusing (see the following link for more information). MapQ scores of 0 and 1 have a special meaning: if a read maps equally well to multiple different locations on a .fasta file, it always gets a MapQ score of 0 or 1.

### How does inStrain compare to other bioinformatics tools for strains analysis?¶

A major difference is inStrain’s use of the popANI and conANI, which allow consideration of minor alleles when performing genomic comparisons. See Important concepts for more information.

### What can inStrain do?¶

inStrain includes calculation of nucleotide diversity, calling SNPs (including non-synonymous and synonymous variants), reporting accurate coverage / breadth, and calculating linkage disequilibrium in the contexts of genomes, contigs, and individual genes.

inStrain also includes comparing the frequencies of fixed and segregating variants between sequenced populations with extremely high accuracy, out-performing other popular strain-resolved metagenomics programs.

The typical use-case is to generate a .bam file by mapping metagenomic reads to a bacterial genome that is present in the metagenomic sample, and using inStrain to characterize the microdiversity present.

Another common use-case is detailed strain comparisons that involve comparing the genetic diversity of two populations and calculating the extent to which they overlap. This allows for the calculation of population ANI values for extremely similar genomic populations (>99.999% average nucleotide identity).

Installation
To get started using the program
Expected output
To view example output
User Manual
For information on how to prepare data for inStrain and run inStrain
Important concepts
For detailed information on how to make sure inStrain is running correctly

### How does inStrain work?¶

The reasoning behind inStrain is that every sequencing read is derived from a single DNA molecule (and thus a single cell) in the original population of a given microbial species. During assembly, the consensus of these reads are assembled into contigs and these contigs are binned into genomes - but by returning to assess the variation in the reads that assembled into the contigs, we can characterize the genetic diversity of the population that contributed to the contigs and genomes.

The basic steps:

1. Map reads to a .fasta file to create a .bam file
3. Calculate nucleotide diversity and SNVs
5. Optional: calculate gene statistics and SNV function
6. Optional: compare SNVs between samples.

### What is unique about the way that inStrain compares strains?¶

Most strain-resolved pipelines compare the dominant allele at each position. If you have two closely related strains A and B in sample 1, with B being at higher abundance, and two closely related strains A and C in sample 2, with C being at higher abundance, most strain comparison pipelines will in actuality compare strain B and C. This is because they work on the principle of finding the dominant strain in each sample and then comparing the dominant strains. InStrain, on the other hand, is able to identify the fact that A is present in both samples. This is because it doesn’t just compare the dominant alleles, but compares all alleles in the two populations. See module_descriptions and choosing_parameters for more information.

### What is a population?¶

To characterize intra-population genetic diversity, it stands to reason that you first require an adequate definition of “population”. InStrain relies mainly on population definitions that are largely technically limited, but also coincide conveniently with possibly biological real microbial population constraints (see Olm et. al. mSystems 2020 and Jain et. al. Nature Communications 2018). Often, we dereplicate genomes from an environment at average nucleotide identities (ANI) from 95% to 99%, depending on the heterogeneity expected within each sample - lower ANIs might be preferred with more complex samples. We then assign reads to each genome’s population by stringently requiring that combined read pairs for SNP calling be properly mapped pairs with an similarity to the consensus of at least 95% by default, so that the cell that the read pair came from was at least 95% similar to the average consensus genotype at that position. Within an environment, inStrain makes it possible to adjust these parameters as needed and builds plots which can be used to estimate the best cutoffs for each project.

### What are inStrain’s computational requirements?¶

The two computational resources to consider when running inStrain are the number of processes given (-p) and the amount of RAM on the computer (usually not adjustable unless using cloud-based computing). Using inStrain v1.3.3, running inStrain on a .bam file of moderate size (1 Gbp of less) will generally take less than an hour with 6 cores, and use about 8Gb of RAM. InStrain is designed to handle large .bam files as well. Running a huge .bam file (30 Gbp) with 32 cores, for example, will take ~2 hours and use about 128Gb of RAM. The more processes you give inStrain the longer it will run, but also the more RAM it will use. See Important concepts for information on reducing compute requirements.

### How can I infer the relative abundance of each strain cluster within the metagenomes?¶

At the moment you can only compare the relative abundance of the populations between samples. Say strain A, based on genome X, is in samples 1 and 2. You now know that genome X is the same strain in both samples, so you could compare the relative abundance of genome X in samples 1 and 2. But if multiple strains are present within genome X, there’s no way to phase them out.

InStrain compare isn’t really phasing out multiple strains in a sample, it’s just seeing if there is micro-diversity overlap between samples. Conceptually inStrain operates on the idea of “strain clouds” more than distinct strains. InStrain isn’t able to tell the number of strains that are shared between two samples either, just that there is population-level overlap for some particular genome. Doing haplotype phasing is something we’ve considered and may add in the future, but the feature won’t be coming any time in the near future.

### How can I determine the relative abundance of detected populations?¶

Relative abundance can be calculated a number of different ways, but the way I like to do it “percentage of reads”. So if your sample has 100 reads, and 15 reads map to genome X, the relative abundance of genome X is 15%. Because inStrain does not know the number of reads per sample, it cannot calculate this metric for you. You have to calculate it yourself by dividing the total reads in the sample by the value filtered_read_pair_count reported in the inStrain genome_wide output.

### What mapping software can be used to generate .bam files for inStrain?¶

Bowtie2 is a common one the works well, but any software that generates .bam files should work. Some mapping software modifies .fasta file headers during mapping (including the tool BBMap and SNAP). Include the flag --use_full_fasta_header when mapping with these programs to properly handle this.