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Welcome to the WAAFLE tutorial

WAAFLE (a Workflow to Annotate Assemblies and Find LGT Events) is a method for identifying novel lateral gene transfer (LGT) events in assembled metagenomic contigs. "Novel" in this context means that the LGT event has not been previously observed in a sequenced isolate genome of the putative recipient species.

You can install WAAFLE from pypi or from source. For additional help with installation and use, see the WAAFLE manual or the WAAFLE channel of the bioBakery support forum.

Table of contents

How does WAAFLE work?

WAAFLE integrates gene sequence homology and taxonomic provenance to identify metagenomic contigs explained by pairs of microbial clades but not by single clades (i.e., putative LGTs). More specifically, for each locus in a contig, WAAFLE identifies the best hit to each species in a pangenome database. WAAFLE then looks for a species whose minimum per-locus score exceeds a lenient homology threshold (k1). If one or more species meet this criterion, then the contig is assigned to the species with the best average score. Otherwise, the process is repeated for pairs of species. If all per-locus scores for a pair of species exceed a stringent homology threshold (k2), then the contig is considered a putative LGT between those species.

Consider the following pair of examples:

Fig. 1

Both cases consider contigs with six protein-coding loci (determined from WAAFLE itself or an independent ORF-calling program such as Prodigal). In Example 1, genes from species C are able to explain all of the loci reasonably well (with scores exceeding k1). Hence, WAAFLE will report this contig as a one-species contig explained by species C.

In Example 2, no single species can explain all of the loci (the minimum score for each species is below k1). However, the pair of species A and B have strong hits (>k2) to all loci, and so WAAFLE concludes that this contig may represent an A+B LGT. Given the AABBAA synteny pattern, a B-to-A transfer would appear to be the more likely mechanism.

Note that in Example 2, if species C had hits to the 2nd and 5th loci that exceeded k1 (as in Example 1), WAAFLE's algorithm would conservatively favor the weaker one-species explanation for the contig rather than invoking a two-species (LGT-based) explanation.

Getting started with WAAFLE

You can test if WAAFLE is available in your computing environment by running waafle_search -h, which should return a help menu. If it doesn't, then please consult the WAAFLE manual for help installing and setting up WAAFLE.

If you cloned the WAAFLE repository, or are working in a bioBakery computing environment, you may already have the WAAFLE demo files available to you. If not, or if you're not sure, you can download them from here. The download will appear as a zip archive, Extract the archive (with e.g. unzip on the command line) and then navigate to waafle-master/demo to find the demo files.

Demo introduction

Under the WAAFLE demo/ folder you'll find three subfolders:

  • input/ contains files used in the tutorial.
  • output/ contains the expected outputs from each tutorial step.
  • output_prodigal/ contains the expected outputs from each tutorial step assuming you used Prodigal for gene calling rather than WAAFLE itself.

The input/ folder contains three pieces of data:

  • demo_contigs.fna is a set of input contigs derived from HMP stool sample SRS011084. (These contigs have been pre-screened for uniform coverage to help rule out misassembly events.)
  • demo_waafledb/ is a reduced, WAAFLE-formatted BLAST database.
  • demo_taxonomy.tsv is a reduced taxonomy file for the species in the BLAST database.

Inspect the demo files with less or other shell commands to answer the following discussion questions.

  • How many contigs are present in the contigs file?
  • How many unique species are present in the taxonomy file? (Hint: the structure of the taxonomy file is a list of tab-separated child-parent relationships; species entries are prefixed with s__.)
  • Which species has the most supporting genomes in the taxonomy file? (Hint: genomes are the leaves of the taxonomy, and are prefixed with t__.)

Step 1. Generate BLAST hits with waafle_search.

The first step in the WAAFLE workflow is to search the input contigs against a WAAFLE-formatted pangenome database. See the options for the waafle_search program using the help command:

waafle_search --help

The two critical parameters are the query (contigs) and database. Let's search the demo contigs (demo_contigs.fna) against the demo database:

waafle_search input/demo_contigs.fna input/demo_waafledb/demo_waafledb

The command will finish quickly as both the input and database are small. By default, this produces an output file demo_contigs.blastout. Let's inspect this output file with less demo_contigs.blastout:

1458  GENE000041992|s__Faecalibacterium_prausnitzii|UniProt=D4JZK5  5194  1347  1347  3675  5021  1347  1     92.72  1249  0   0.0     1947  minus
1458  GENE000042498|s__Faecalibacterium_prausnitzii|UniProt=D4JZK7  5194  1440  1407  830   2236  1     1407  87.21  1227  0   0.0     1602  plus
1458  GENE000041989|s__Faecalibacterium_prausnitzii|UniProt=D4JZK5  5194  1347  1350  3675  5021  1347  1     87.56  1182  6   0.0     1559  minus
1458  GENE000041988|s__Faecalibacterium_prausnitzii|UniProt=D4JZK5  5194  1347  1346  3677  5021  1345  1     87.44  1177  2   0.0     1550  minus
1458  GENE000043174|s__Faecalibacterium_prausnitzii|UniProt=D4JZK6  5194  1281  1283  2349  3626  1281  1     87.45  1122  7   0.0     1478  minus
1458  GENE000042009|s__Faecalibacterium_prausnitzii|UniProt=None    5194  1347  1341  3682  5021  1340  1     86.43  1159  2   0.0     1469  minus
1458  GENE000042343|s__Faecalibacterium_prausnitzii|UniProt=C7H5K2  5194  1434  1377  864   2237  35    1408  82.64  1138  6   0.0     1214  plus
1458  GENE000042322|s__Faecalibacterium_prausnitzii|UniProt=D4K630  5194  1431  1395  850   2237  21    1408  80.72  1126  14  0.0     1074  plus
1458  GENE000042865|s__Faecalibacterium_prausnitzii|UniProt=C7H5K3  5194  1212  654   2415  3065  1206  556   84.56  553   6   0.0     643   minus
1458  GENE000038197|s__Faecalibacterium_prausnitzii|UniProt=D4JZK8  5194  588   575   106   679   1     574   85.57  492   2   4e-170  601   plus

The columns of the output match the requested columns from the BLAST command. Most critically, the first and second columns provide a mapping from the input contigs (query sequences) to genes in the demo database (subject sequences). Each subject sequence has the following format:


In the demo database, genes have been annotated with UniProt accession numbers. You can look up individual accession numbers on the UniProt website: e.g.

Answer the following questions about the BLAST output by using shell commands or visual inspection:

  • Which contig received the most BLAST hits?
  • Did any contigs receive hits to more than one species?

Step 2. Call genes with waafle_genecaller.

In order to classify the contigs, WAAFLE compares the BLAST hits generated above to a set of predicted protein-coding loci within the contigs, as defined by a GFF file. WAAFLE includes a utility to call genes within contigs based on the BLAST output itself by clustering the start and stop coordinates of hits along the length of the contig.

waafle_genecaller --help

This utility requires a single input to run: the BLAST output file:

waafle_genecaller demo_contigs.blastout

This produced a file in GFF format called demo_contigs.gff. Inspect its contents using the less command:

1458  waafle_genecaller  gene  106   679   .  +  0  .
1458  waafle_genecaller  gene  830   2237  .  +  0  .
1458  waafle_genecaller  gene  2349  3626  .  -  0  .
1458  waafle_genecaller  gene  3675  5021  .  -  0  .
1535  waafle_genecaller  gene  975   1992  .  -  0  .
1535  waafle_genecaller  gene  2041  3363  .  -  0  .
1535  waafle_genecaller  gene  3388  4125  .  -  0  .
1535  waafle_genecaller  gene  4486  6028  .  +  0  .
1689  waafle_genecaller  gene  335   1245  .  +  0  .
1689  waafle_genecaller  gene  1798  2779  .  -  0  .

Columns 1, 4, and 5 are the most important: they provide an index of the gene start and stop coordinates within each contig.

Answer the following questions about the GFF output by using shell commands or visual inspection:

  • Which contig contains the most predicted genes?
  • Are the contigs 'gene-dense'? Does this match your expectation for prokaryotic genomes?

Step 3. Find LGT-containing contigs with waafle_orgscorer.

The last step in the WAAFLE workflow is also the most important: comparing per-species BLAST hits with the contig's gene coordinates (loci) to try to find one- and two-species explanations for contigs (as described in the algorithm overview above). This step is peformed by the waafle_orgscorer utility. This utility has many tunable parameters, most of which are devoted to filtering and formatting the outputs.

You can inspect the parameters of waafle_orgscorer using the flag -h (for a summary) or --help (for details):

usage: [-h] [--outdir <path>] [--basename <str>]
                           [--write-details] [--quiet] [-k1 <0.0-1.0>]
                           [-k2 <0.0-1.0>]
                           [--disambiguate-one <report-best/meld>]
                           [--disambiguate-two <report-best/jump/meld>]
                           [--range <float>] [--jump-taxonomy <1-N>]
                           [--allow-lca] [--ambiguous-fraction <0.0-1.0>]
                           [--clade-genes <1-N>] [--clade-leaves <1-N>]
                           [--sister-penalty <0.0-1.0>]
                           [--weak-loci <ignore/penalize/assign-unknown>]
                           [--transfer-annotations <lenient/strict/very-strict>]
                           [--min-overlap <0.0-1.0>] [--min-gene-length <int>]
                           [--min-scov <float>] [--stranded]
                           contigs blastout gff taxonomy

The two most important parameters are k1 and k2, as introduced in the algorithm summary above.

  • What are the default values of k1 and k2?

Lets try a run of waafle_orgscorer with only the four required arguments, contigs blastout gff taxonomy:

waafle_orgscorer \
	input/demo_contigs.fna \
	demo_contigs.blastout \
	demo_contigs.gff \

This produces three output files:

Examining one-clade (no-LGT) contigs

Most contigs are assigned to the no_lgt bin. Let's inspect a subset of the fields from this file with cut and less:

cut -f1,4-7 demo_contigs.no_lgt.tsv | less

Which yields:

14237        0.983      0.989      AAAA       s__Faecalibacterium_prausnitzii
14258        0.950      0.994      AAAAAAAAA  s__Eubacterium_rectale
14270        0.992      0.995      AAAA       s__Roseburia_intestinalis
14274        0.833      0.913      AAAA       s__Faecalibacterium_prausnitzii
14307        0.730      0.870      AAAA       s__Faecalibacterium_prausnitzii
14339        0.997      0.999      AAAAAA     s__Roseburia_intestinalis
14449        0.818      0.968      AAAAAA     s__Faecalibacterium_prausnitzii
14496        0.815      0.867      AAAA       s__Faecalibacterium_prausnitzii
14528        0.901      0.917      AAAA       s__Collinsella_aerofaciens

In the case of the first contig, 14237, these fields tell us that the contig was best explained by Faecalibacterium prausnitzii. The contig had four genes (evident from the AAAA synteny). F. prausnitzii had a minimum score over these genes of 0.983 (much greater than the threshold of 0.5), and its average score was similarly high at 0.989. We are very confident that this contig represents a fragment of F. prausnitzii genome.

Answer the following questions about the one-species contigs by using shell commands, visual inspection, or internet research:

  • Are the species detected reasonable for a human gut sample?
  • Which species contributed the most contigs to the metagenomic assembly?
  • Which taxonomic assignment was WAAFLE least confident about?

When WAAFLE fails to find a one- or two-species explanation, it repeats its search at the next highest-level clades, looking for (e.g.) one-genus vs. two-genera explanations.

  • Are there any instances of this behavior in the no_lgt output?

Examining two-clade (putative LGT) contigs

Now for exciting part: examining the putative LGTs in the demo_contigs.lgt.tsv file. We'll again focus on a subset of the output columns:

cut -f1,4-10 demo_contigs.lgt.tsv

Which yields:

CONTIG_NAME  MIN_MAX_SCORE  AVG_MAX_SCORE  SYNTENY       DIRECTION  CLADE_A                     CLADE_B                          LCA
12571        0.856          0.965          AABAAAA       B>A        s__Ruminococcus_bromii      s__Faecalibacterium_prausnitzii  f__Ruminococcaceae

In the case of the first contig, 12571, these fields tell us that the contig was best explained by a putative LGT between Ruminococcus bromii and Faecalibacterium prausnitzii: two species that are related at the family level [according to the lowest common ancestor (LCA) field]. The synteny pattern AABAAAA suggests that a single F. prausnitzii gene (B) inserted into the R. bromii genome.

The min-max score entry indicates that, across the seven loci of this contig, one of these species always scored at least 0.856 (this exceeded the default k2 value of 0.8, allowing the LGT to be called).

Add column 16 to the cut command above to inspect the annotations of these genes:


By default, WAAFLE assigns the annotation of the best BLAST hit at each locus. Here, the best hit to the sixth |-delimitted was not annotated in UniProt (it receives a None annotation).

Answer the following questions about the two-species contigs by using shell commands, visual inspection, or internet research:

  • What is the function of the LGT'ed gene in the example above?
  • What is the most remote LGT event (i.e. the event with the highest-level LCA)?
  • Are there any LGT contigs where the donor and recipient cannot be determined confidently?

Challenge questions:

  • Which species is the most frequent LGT donor?
  • Do any species appear as LGT donors but never as recipients?
  • How many LGT events occurred per 1,000 assembled genes?

Extension A: Working with Prodigal gene calls

In the workflow above, we used waafle_genecaller to identify potential coding loci in our contigs. As alluded to above, we can also perform this step with an independent open reading frame (ORF) detection system, such as Prodigal.

Run prodigal on the input contigs to produce an alternate GFF file:

prodigal.linux \
    -i input/demo_contigs.fna \
    -f gff \
    -o demo_contigs.prodigal.gff

(If you don't have Prodigal available on your system, you can use the Prodigal GFF file in output_prodigal/.) Inspect the alternate GFF file demo_contigs.prodigal.gff with less. You'll find it has more details than the equivalent file produced by WAAFLE, but the overall format is the same.

Repeat Step 3 above using the alternate GFF file and adding the argument --basename demo_contigs.prodigal to the waafle_orgscorer call (this will prevent overwriting the original outputs). Inspect the outputs.

You'll notice that the synteny strings now contain a ~ character. This corresponds to a locus that never received a "good" hit to any species (i.e. with homology score >k1). By default, waafle_orgscorer will ignore such loci. You can change this behavior with the --weak-loci flag.

  • How do the results change using the Prodigal GFF file?
  • What are pros and cons of using an ORF-based gene caller rather than homology-based gene definitions?

Extension B: Experimenting with LGT-calling parameters

As noted above, there are many options for tuning the behavior of waafle_orgscorer using its configuration flags. The default settings for these parameters have all been pre-tuned for high sensitivity and specificity (based on evaluations of synthetic contigs of known LGT status). However, depending on your application, it may be useful to tune parameters for a more sensitive and less specific analysis (or vice versa).

Raising k1

Repeat the original analyis from Step 3 above, but set k1 to 0.8 instead of the default of 0.5 (i.e. so the k1 parameter is NOT more lenient than k2.)

  • What effect does a higher k1 have?
  • Does this make the analysis more or less sensitive/specific?

Starting with genera

Perform a run adding the parameter --jump-taxonomy 1. This will begin the analysis using genus-level clades rather than species-level clades.

  • What effect does starting at the genus level have?
  • Does this make the analysis more or less sensitive/specific?

Requiring more isolate genome support

Perform a run adding --clade-leaves 2. This will require clades in a two-species contig to be supported by at least two isolate genomes.

  • What effect does requiring more supporting isolate genomes have on reported LGTs?
  • What is a potential concern when describing a LGT in which the donor is supported by a single genome?