software

Global genomic framework for typhoid

It’s been over a year since we published the first global whole-genome snapshot of nearly 2000 genomes of the typhoid bacterium, Salmonella Typhi in Nature Genetics.

That paper focused on the emergence and global dissemination of what we’ve been calling for years the “H58” clone (see this blog post). This clone accounted for nearly half of all the isolates sequenced, and is a big deal because it tends to be multidrug resistant (MDR), carrying a suite of resistance genes that render all the cheap, first-line drugs like chloramphenicol, ampicillin, and trimethoprim-sufamethoxazole useless for treatment. Detailed genomic epi studies show the local impact of the arrival of MDR H58 in countries as widespread as Malawi and Cambodia; and the emergence of fluoroquinolone resistant H58 sublineage in India and Nepal recently stopped a treatment trial because the current standard of care – ciprofloxacin – was resulting in frequent treatment failure.

While H58 is important, the global Typhi population contains a lot of genomic diversity outside the H58 clone, and we’ve turned our attention to the rest of the population now in a new paper in Nature Communications: “An extended genotyping framework for Salmonella enterica serovar Typhi, the cause of human typhoid

First, we decided that we needed to revisit the haplotyping scheme of Roumagnac et al (from which H58 gets its name), which was based on just ~80 genes, using the whole genome phylogeny. Here is the tree inferred from core genome SNPs in 1832 Typhi strains, with the old haplotypes indicated by the coloured ring around the outside. It’s pretty easy to see that some haplotypes (like H52 and H1) actually comprise multiple distinct phylogenetic lineages (low resolution), while others subdivide lineages (excessive resolution).

FigS1_GlobalTreeColouredByHgroups

Whole genome SNP tree for 1832 strains, outer ring indicates haplotypes based on mutations in 80 genes as defined in Roumagnac et al, Science 2006.

We used BAPS to define genetic clusters at various levels (thanks to Tom Connor for running this). We settled on 3 levels of hierarchical clustering, indicated in the tree below:

• 4 nested primary clusters (inner-most ring; yellow, green, blue, red). These have 100% bootstrap support and are each characterised by >20 SNPs

• Clusters are further divided into 16 clades (middle ring and labels). The median pairwise distance between isolates in the same clade is 109 SNPs, while the inter-clade SNP distance averages 243 SNPs.

• Clades are further divided into 49 subclades, indicated by alternating background shading colours. The median pairwise distance between isolates in the same subclade is 25 SNPs.

Fig1_noH58_clade_colours_subcladebg_190515_labelled_ED.png

Tree indicating new phylo-informed genotypes. Primary clusters 1-4 are indicated in the inner ring. Branch colours indicate clades, which are also labelled on the outside and coloured in the outer ring. Subclades are indicated by alternating background shading.

subcladerectOne of the key reasons we wanted to define the phylogenetic lineages in this way is to make them easier to identify and talk about. I’ve always been a fan of MLST for this reason, since it’s much easier to talk about K. pneumoniae ST258, ST11, ST15 etc than ‘that lineage that has reference strain X in it’. So we introduce a hierarchical nomenclature system, similar to the one currently in use for Mycobacterium tuberculosis, where the 4 primary clusters (1, 2, 3, 4) are subdivided into 16 clades (1.1, 1.2; 2.1, 2.2, etc) which in turn are subdivided into 49 subclades (1.1.1, 1.1.2, etc). This has the advantage of conveying hierarchical relationships between groups – e.g. 2.2.1 and 2.2.2 are sister subclades within clade 2.2, which is a sister clade of 2.1.

The subclades are easier to distinguish in the collapsed rectangular tree on the right, where each subclade is represented by just one strain.

Some BAPS clusters were polyphyletic and consisted of isolates belonging to rare phylogenetic lineages whose common ancestor in the tree coincided with the common ancestor of an entire clade (n=9) or primary cluster (n=2). These groups contain isolates that, given increased numbers, may emerge as distinct clusters that form sister taxa within the parent clade (or primary cluster), and were given the suffix ‘.0’ rather than a defined cluster number (e.g. 3.0 or 3.1.0) to indicate non-equivalence with the properly differentiated sister clades (n=16) or subclades (n=49). As more genomes are added, these are expected to be more clearly differented into distinct groups and given proper clade/subclade designations.

Next we defined a set of 68 SNPs that can be used to genotype isolates into these groups. We chose one SNP for each primary cluster, clade and subclade (preferentially choosing intragenic SNPs in well-conserved core genes). The SNPs are detailed in a supplementary spreadsheet, and we provide a script to assign strains to genotypes based on an input BAM or VCF file generated by mapping to the reference genome for Typhi strain CT18.

An isolate that belongs to a differentiated subclade such as 2.1.4 will be hierarchically identified by carrying the derived allele for primary cluster 2 (but not the nested clusters 3 and 4); the derived allele for clade 2.1 (but no other clades) and the derived allele for subclade 2.1.4 (but no other subclades). It is possible for an isolate to carry derived alleles for a primary cluster and clade with no further differentiation into subclade.

The clone formally known as H58
Under the new scheme, the infamous H58 clone is named subclade 4.3.1, which so far has no sister clades. I suspect those of us familiar with Typhi population genomics will keep referring to it informally as H58 for some time, since that name is now well known… but I will try to re-train myself to call it 4.3.1 (H58).

Now the fun part: exploring the geographical distribution of these lineages.

Fig1c_worldmap_pies_subclades

Figure 1c from the paper. Pie colours indicate clades found in each WHO region in the global data set (key is in the tree figure above).

In the paper we go on to show that:
• clades are widely geographically distributed, while subclades are geographically constrained (see heatmap below);
• genotyping can be used to predict the geographical origin of travel-associated typhoid in patients in London;
• even better predictions can be obtained based on genome-wide SNP distances to our reference panel of >1800 isolates… but of course that involves a lot more computationally intensive comparisons than a quick screen of a new isolate’s BAM file.

Screen Shot 2016-08-29 at 11.47.15 pm.png

Figure 2 of the paper, showing the geographical distribution of subclades, which shows most subclades are restricted to a single region. For this analysis, the effect of local outbreaks has been minimised by replacing groups of strains that share the same subclade and year and country of isolation with a single representative strain.

You can read the full details in the paper, but here I just want to highlight that you can now explore the global genomic framework for Typhi – including genotype designations as well as temporal and geographic data – interactively in MicroReact.

tree.png

 

How does genotyping help with studying local populations?

We have already begun using the new genotyping scheme in local typhoid studies. I find this a really helpful way to describe/summarise the local populations, and place them in the context of the global population without resorting to large trees.

For example in this recent Nigerian study, we described the population like this: “The majority of isolates (84/128, 66%) belonged to genotype 3.1.1 , which is relatively common across Africa, predominantly western and central countries. In the wider African collection genotype 3.1.1 was represented by isolates from neighbouring Cameroon and across West Africa (Benin, Togo, Ivory Coast, Burkina Faso, Mali, Guinea and Mauritania) suggesting long-term inter-country exchange within the region. Most of the remaining isolates belonged to four other genotypes (4.1, 2.2, 2.3.1 and 0.0.3).”

Of course genotype assignment is not the end of the story – we still want to build whole-genome trees to explore the relationships of local isolates with those from other countries. Importantly, working with genotypes means that we can achieve this without needing to build a megatree of all isolates in the local + global collections (n>2000). Instead, we can use the genotypes to identify which strains from the global collection are relatives of the Nigerian isolates, and build a much smaller tree that still captures all of the information about transmission/transfer between Nigeria and other countries:

journal-pntd-0004781-g001

The tree and map were made using MicroReact, you can recreate theme here: http://microreact.org/project/styphi_nigeria To get this colour scheme just click on the eye icon (bottom left) and select ‘country’; and to get the fan style tree, click the settings button (top right) and click the fan shape.

Another example is in our recent paper on isolates collected in Thailand before and after the introduction of their national vaccination program (pre-print here):

  • Genotype 3.2.1 was the most common (n=14, 32%), followed by genotype 2.1.7 (n=10, 23%)
  • Genotypes 2.0 (n=1, 2%) and 4.1 (n=3, 7%) were observed only in 1973 (pre-vaccine period)
  • Genotypes 2.1.7 (n=10, 23%), 2.3.4 (n=1, 2%), 3.4.0 (n=2, 5%), 3.0.0 (n=3, 7%), 3.1.2 (n=2, 5%), were observed only after 1981 (post-vaccine period)
  • Genotypes 3.2.1 and 2.4.0 were observed amongst both pre- and post-vaccine isolates, but the subclade phylogenies show that these more likely to represent re-introduction of strains from neighbouring countries than persistence within Thailand throughout the immunisation program.
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Microbial Genomics methods

MGen (Microbial Genomics) was created last year by the UK’s Microbiology Society, with the aim of becoming the go-to journal for microbial genomics research. As a Senior Editor, I was asked to help mark the occasion of MGen’s six month anniversary (on Jan 15, 2016), by reviewing the 24 papers published in the journal’s first six months since launch.
The full review is is available over on the MGen site, but I wanted to draw particular attention here to trends in the analysis tools used by the genomic epidemiology papers published so far in MGen, which largely reflects what’s been happening across the field generally.

tools word cloud

One third of the articles are cutting edge genomic epidemiologyin action, using genomics to investigate the evolution and transmission of a range of pathogens, from anthrax to dysentery and food poisoning.

Interestingly all of these studies used similar methodologies, reflecting the maturity of this field of research.

Common approaches include: (i) sequencing large numbers of isolates using high-throughput Illumina platforms; (ii) the identification of SNPs (single nucleotide polymorphisms) using read mapping approaches (with BWA, SMALT, SAMtools and GATK being popular tools); and (iii) uniform use of RAxML for generating maximum likelihood phylogenies.

Some also used BEAST to estimate mutation rates, divergence dates and phylogeographical patterns. Interestingly, half of these papers utilised MLST (multi-locus sequence typing) to identify clades, showing that this sub-genomic approach based on capillary sequencing of ~7 gene fragments is still considered useful by many genomic researchers. [NOTE: nice to see that most who were inferring MLST from Illumina data were using our SRST or SRST2 software.]

Almost all of the genomic epidemiology studies took steps to remove SNPs introduced via recombination, in order to capture the underlying signals of vertical inheritance that are so important for transmission studies. Popular tools were BratNextGen, Gubbins and ClonalFrameML, which were all published within the last 3 years.

For pan genome analysis, Velvet and SPAdes were the most popular tools for bacterial genome assembly, with Prokka and Prodigal for gene annotation, and LS-BSR and related approaches being commonly used to cluster orthologous groups of genes.

 

Most of this won’t be any surprise to people working in bacterial genomic epi, but I think it’s great to see that consensus is emerging on how best to do these sorts of analyses, and to at last have some reliable tools for detecting and accounting for recombination.

The area of least agreement remains SNP calling – which mainly comes down to which read mappers to use, and which SNP calling algorithms and filtering to go with? This is a complex area, as highlighted in the recent review “Best practices for evaluating single nucleotide variant calling methods for microbial genomics” in Frontiers in Genetics, which did a very thorough job of examining the issues that need to be considered, but (quite deliberately) doesn’t provide an answer to “which tool is best?”.

Although there is still no real consensus on exact methods for SNP calling, I think most of the tools people are using (ie a good, stable read mapper followed by SNP calling with an established tool like SAMtools or GATK, with some basic filtering to remove low-evidence or ambiguous calls) end up with very similar answers (as we saw with the NGS outbreak analysis challenge session held at the ASM NGS meeting in September 2015). All in all it seems to me that the use of genomics for public health & diagnostic microbiology is in far better shape in this respect than clinical human genomics, which is going through something of a crisis involving wide discrepancies in variant calling as well as uncertainty around data interpretation.

Tracking down insertion sequences causing polymixin resistance in Acinetobacter baumannii

The first plasmid-borne colistin resistance gene was reported late last year. This was big news, but the vast majority of clinically relevant resistance to colistin and related polymixin drugs, which arises frequently in human patients being treated with these drugs, is due to de novo mutations in chromosomal genes. This has been studied quite a lot recently in Acinetobacter baumannii and Klebsiella pneumoniae, where colistin is frequently used to treat patients who are infected with carbapanem resistant strains that are also resistant to pretty much all the other antibiotics as well.

There have been quite a few studies looking for the causative mutations that underly colistin resistance in Acinetobacter and Klebsiella, by comparing the genomes of resistant and susceptible forms of the ‘same strain’ sequenced with high throughput, short read sequencing platforms like Illumina. The typical approach is to catalogue all the differences between strains and look for SNPs and other differences between them. However colistin resistance is usually associated with upregulating pmr activity via point mutations or inactivating the regulator mgrB, or by inactivating the lpx cluster in Acinetobacter. A very common way for gene inactivation to occur is by an insertion sequence (IS) hopping into the open reading frame and disrupting it. IS insertions can also cause resistance by upregulating the expression of intrinsic efflux pump or beta-lactamase genes, for example the ampC gene in A. baumannii. These insertions can be tricky to find using short read data, and are sometimes missed by regular mapping or assembly based approaches.

Luckily we now have two great tools for tracking down such mutations – ISMapper and Bandage – which were critical to tracking down polymixin resistance mutations in a recent study of A. baumannii with colleagues in Singapore. The paper is published in Antimicrobial Agents and Chemotherapy, but unfortunately is paywalled… so quick summary: basically, our clinical researcher friends in Singapore (hello Li Yang!) had 10 pairs of A. baumannii isolates, consisting of a susceptible parent isolate and a derived polymixin isolate (2 that evolved in vivo during treatment with polymixins, and 8 that evolved in vitro during polymixin exposure).

Simply screening for point mutations and deletions identified causative mutations in pmr and/or lpx clusters for 8/10 genomes, but two remained unexplained. A quick screen of the genome assemblies for IS using ISfinder identified several different IS that were present in the 10 A. baumannii genomes. However as these tend to be multi-copy in the genome, they were mostly separated into their own contigs in the assemblies. Enter ISMapper and Bandage, two open source software packages from Jane Hawkey and Ryan Wick in my lab.

First we used ISMapper to identify the locations of all IS sequences in each genome… this involves passing ISMapper each of the different IS sequences, a reference genome to compare to, and the Illumina read sets for the various strains. The isolates were all from global clones 1 or 2 (GC1, GC2), so to get the best results we used a GC1 reference genome for typing IS in the GC1 strains, and a GC2 reference for the GC2 strains. A quick tabulation of the results reveals all the locations of the various IS in each sequence. This identified differential IS insertions in lpx genes (lpxA, lpxC) in three strain pairs, including one in which no other causative mutations had been identified. There was also an IS15 insertion in the mutS gene in one isolate that had many more SNPs and deletions than the others.

ISMapper results

ISMapper results for GC2 strain pairs, showing IS that differed within susceptible-resistant pairs.

ISMapper results for GC1

ISMapper results for GC1 strain pairs, showing IS that differed within susceptible-resistant pairs.

Cool! But what about that one last pair, where ISMapper didn’t find any differences at all between the resistant and susceptible read sets? This time we turned to Bandage, to inspect the genome assemblies and see if we could find a smoking gun. Now we had a clear hypothesis too – we were looking explicitly for interruptions in lpx genesSo we created new assemblies for these read sets using SPAdes. We loaded the graph of the susceptible isolate in Bandage first, and used the inbuilt BLAST search to locate the lpx genes within the graph – all were intact as expected, sitting happily in the middle of long contigs.

Bandage screenshot

Contig containing the lpxC gene (blue) in SPAdes assembly of the susceptible isolate

Then we loaded the graph of the polymixin resistant isolate in Bandage first, and did the BLAST searches. The pmrB locus was intact, but the lpxC gene was interrupted. Very interrupted! No wonder ISMapper didn’t find this as it’s not an IS insertion at all, but rather the gene is interrupted by a large sequence, in an event that appears to involved the translocation of the entire genomic resistance island AbaR4 into the middle of the lpxC open reading frame.

Bandage screenshot

Interruption of lpxC associated with movement of the antibiotic resistance related genomic island, AbaR

The above image is created by doing BLAST searches (within Bandage) for the lpxC gene, AbaR4 gene and ISAba1 gene like this…

Bandage Screen Shot

BLAST search within an assembly graph using Bandage

…and then selecting ‘BLAST hits (solid)’ under the ‘Graph display’ settings on the left hand side of the Bandage viewer.

Screen Shot 2016-01-12 at 6.58.17 pm

 

Oh and just in case you think this is a weird one-off event that maybe you don’t need to worry about in your own genome data… check out the recent report from Scott Beaston and David Paterson in Queensland, who sequenced a nasty Klebsiella strain that was resistant to everything under the sun including carbapenems and colistin. They sequenced the genome with PacBio, and found the ISEcp1blaOXA-181 mobile element (which confers resistance to carbapenems) inserted into the mgrB regulatory gene in the chromosome, whose inactivation is responsible for colistin resistance. Oh and they also found another mobile element, ISEcp1blaCTX-M-15, inserted into the gene ompK35. Guess what inactivating this gene gives you? Cephalosporin resistance.

 

SEE ALSO: this post on using ISMapper and Bandage to track down multidrug resistance in Salmonella Typhi, the causative agent of typhoid

Locating drug resistance regions in short reads, using Bandage and ISMapper

In our recent paper on Salmonella Typhi, we described the multidrug resistant (MDR) clone H58 that is sweeping the world.

We’ve been monitoring this clone for almost 10 years (and the data suggests it actually emerged in the early 1990s), but the big change noted recently is the movement of the drug resistance locus out of the large conjugative (IncHI1) plasmid and into the chromosome.

I think this is critically important, because it means the large plasmid that carried the multidrug resistance genes into the cell can be lost, relieving the bacteria of the burden associated with replicating and expressing the plasmid genome, without losing multidrug resistance.

However, figuring out the location of the MDR locus is tricky, because it is flanked by copies of the IS1 transposase. This is what it looks like (the red genes on the ends are the IS1 copies).

Multidrug resistance locus in Salmonella Typhi

Multidrug resistance locus in Salmonella Typhi

Repeated sequences like these flanking IS1 sequences complicate assemblies… because there are multiple possible paths in and out of the repeated sequence, most assemblers will place the repeated sequence in its own contig, and the various paths in and out of it in separate contigs.

Here’s an example of an assembly graph showing the MDR locus above, visualised using Bandage, developed by Ryan Wick in my group… The green bit is a BLAST hit to the whole IS1 transposase, and you can see there are multiple alternative paths through the IS:

bandage IS1

And here is the same graph, but this time colouring in all the genes encoded within the MDR locus (BLAST hits to each open reading frame are shown in a different colour; the IS1 has 2 ORFs):

Screen Shot 2015-07-20 at 3.33.59 pm

So, inferring the location of the MDR locus from short read data is important but tricky. What do do?

Well, turns out it’s pretty easy to figure out what’s happening using Bandage! In this example, we can’t tell where the IS1 (and MDR locus) is inserted by looking at the assembled contigs. But by looking at this graph, we can see there are only two paths out of the IS1; one leads into the MDR locus (and back to the IS1), and the other leads to a single contig. By exporting that contig sequence from Bandage and blasting it in NCBI, it’s pretty trivial to discover that this is a piece of Typhi chromosome!

Here’s an example assembly graph from a different strain:
Screen Shot 2015-07-20 at 3.45.54 pmWhen I use Bandage to blast for the IncHI1 plasmid sequence (hits in blue) as well as the MDR locus genes, it’s pretty easy to see that the MDR locus is located in the plasmid:

Screen Shot 2015-07-20 at 3.47.05 pm

So basically – Bandage can be super useful for locating resistance genes (or any genes of interest really), in the presence of repeat sequences!

In the Typhi paper, we had hundreds of genomes to analyse so of course didn’t manually inspect each graph. Instead we used ISMapper (from my PhD student Jane Hawkey) to determine the IS1 insertion sites in each strain, and coupled this with what we knew about the presence of the plasmid and resistance genes in each strain (using SRST2, also from our group), to figure out which strains had the MDR locus and where:

MDR_summary_regionAsRing

Of course, all this is easy with long read sequencing… in the paper we used PacBio to confirm some the insertion sites in cyaA and yidA, and the guys at Public Health England independently found the yidA site using Nanopore.

But with the right tools, you can actually figure out a lot of these questions using Illumina data alone.

R code to infer tree from ANDI output

Just playing with ANDI, for building whole genome trees rapidly from large sets of genome assemblies
Code: https://github.com/evolbioinf/andi/
Paper: http://bioinformatics.oxfordjournals.org/content/31/8/1169

It seems really great, and lightning fast (few seconds on ~100 genomes).

The output is a phylip distance file, which is not something I normally work with, having spent the last 5+ years inferring trees with ML from DNA alignments and not thinking much about distance based methods!

So I wanted to figure out how to handle this output simply in R.

The answer is easy, code below… the only tricky thing is that there were a couple of genomes in my genome set that returned NaN for pairwise distances with other genomes, which need to be removed before you can infer a tree.

Note there are lots more tree inference options in the ape library, nj is just one example.


# function to remove rows with unknowns 
dropNA <- function(d) {
na <- apply(d,1,function(x){sum(x=="NaN")})
while (max(na)>0) {
na.drop <- c(1:length(na))[na==max(na)]
d <- d[-na.drop,-na.drop]
na <- apply(d,1,function(x){sum(x=="NaN")})
}
d
}

# read distance file output by ANDI
d<-read.table("andi.phy",header=F,skip=1,row.names=1)
colnames(d) <- rownames(d)

# remove rows with unknowns
d <- dropNA(d)

# infer tree from distance
t<-nj(as.dist(d))

# write tree
write.tree(t,file="tree.nwk")