Lineage calling

One thing you might want to do with metagenomics data is to identify the species present in your sample. Here we will use SLIMM which is a binning based classifier similar to Kraken.

The reason we often use SLIMM is that it generates cleaned species-level coverage maps as a side effect which we use extensively.

library(mbtools)
## Also loading:
##   - dada2=1.12.1
##   - data.table=1.12.6
##   - ggplot2=3.2.1
##   - magrittr=1.5
##   - phyloseq=1.28.0
##   - ShortRead=1.42.0
##   - yaml=2.2.0
## Found tools:
##   - minimap2=2.17-r941
##   - slimm=0.3.4
##   - samtools=1.9
## 
## Attaching package: 'mbtools'
## The following object is masked _by_ 'package:BiocGenerics':
## 
##     normalize
## The following object is masked from 'package:graphics':
## 
##     layout

Getting the SLIMM DB

We will use our shotgun data again since the reference genomes are also included in the SLIMM database. For real data you will want to align against the full SLIMM database of ~15K genomes.

Download instructions will come soon :/

For now we will only use the SLIMM taxonomy mapping database which is delivered with mbtools as well.

slimm_db <- system.file("extdata/ABVF_SP_CMP_genomes.sldb",
                        package = "mbtools")

which will save the db in the folder refs.

Aligning shotgun reads

One advantage of SLIMM is that it is relatively agnostic to the alignment program. So we can use our normal alignment workflow.

fi <- system.file("extdata/shotgun", package = "mbtools") %>%
      find_read_files()
ref <- system.file("extdata/genomes/zymo_mock.fna.gz",
                   package = "mbtools")

alns <- align_short_reads(
    fi,
    threads = 3,
    reference = ref,
    use_existing = FALSE)
## INFO [2019-11-05 08:20:31] Aligning 3 samples on 3 threads. Keeping up to 100 secondary alignments.
## INFO [2019-11-05 08:20:33] Finished aligning even1.
## INFO [2019-11-05 08:20:35] Finished aligning even2.
## INFO [2019-11-05 08:20:37] Finished aligning even3.

Using SLIMM

SLIMM is again wrapped by a workflow so we can use it passing in an alignment artifact and a configuration.

config <- config_slimm(
    threads = 3,
    database = slimm_db
)

config
## $reports
## NULL
## 
## $bin_width
## [1] 1000
## 
## $relative_cutoff
## [1] 0
## 
## $database
## [1] "/Library/Frameworks/R.framework/Versions/3.6/Resources/library/mbtools/extdata/ABVF_SP_CMP_genomes.sldb"
## 
## $threads
## [1] 3
## 
## $rank
## [1] "species"
## 
## attr(,"class")
## [1] "config"

With this we can perform the SLIMM workflow.

sl <- slimm(alns, config)
## INFO [2019-11-05 08:20:38] running SLIMM on 3 alignments with database /Library/Frameworks/R.framework/Versions/3.6/Resources/library/mbtools/extdata/ABVF_SP_CMP_genomes.sldb.
## INFO [2019-11-05 08:20:38] Parsing abundances on rank `species`.
## INFO [2019-11-05 08:20:38] Fixing taxa names.
## INFO [2019-11-05 08:20:38] Estimating read lengths from a sample of 100 reads per alignment.
## INFO [2019-11-05 08:20:38] Estimated median read length is 100, range is [100, 100].
## INFO [2019-11-05 08:20:38] Parsing coverage profiles with bin width of 1000bp.
## INFO [2019-11-05 08:20:38] Fixing taxa names.

The artifact now contains the species level lineage calls…

sl$abundance
##       rank taxa_id relative reads    id              kingdom
## 1: species      0*        1 12598 even1 unknown_superkingdom
## 2: species      0*        1 12127 even2 unknown_superkingdom
## 3: species      0*        1 12346 even3 unknown_superkingdom
##            phylum         class         order         family genus species
## 1: unknown_phylum unknown_class unknown_order unknown_family  <NA>    <NA>
## 2: unknown_phylum unknown_class unknown_order unknown_family  <NA>    <NA>
## 3: unknown_phylum unknown_class unknown_order unknown_family  <NA>    <NA>

Where we see no successful calls since our coverage is way to low for the example data.

However we can investigate which strains the data aligned to.

sl$coverage[, uniqueN(strain)] %>% print()
## [1] 10
sl$coverage
##     bin_width           reads  genbank
##  1:      1000 0,0,0,0,0,0,... BK006935
##  2:      1000 0,0,0,0,0,0,... AE016830
##  3:      1000 0,0,0,1,0,0,... AE017341
##  4:      1000 2,2,0,0,0,0,... AL009126
##  5:      1000 0,0,0,0,0,0,... AE006468
##  6:      1000 0,2,0,0,0,0,... BA000007
##  7:      1000 0,0,0,0,0,0,... AP008937
##  8:      1000 2,0,2,0,0,0,... AL591824
##  9:      1000 0,0,0,0,0,0,... CP000253
## 10:      1000 0,2,0,0,0,0,... AE004091
## 11:      1000 0,0,0,0,0,0,... BK006935
## 12:      1000 0,0,0,0,0,2,... AE016830
## 13:      1000 0,0,0,0,0,0,... AE017341
## 14:      1000 0,0,0,0,0,0,... AL009126
## 15:      1000 0,0,0,0,4,0,... AE006468
## 16:      1000 0,0,0,0,0,2,... BA000007
## 17:      1000 0,0,0,0,0,0,... AP008937
## 18:      1000 0,0,0,0,0,0,... AL591824
## 19:      1000 2,0,0,0,0,3,... CP000253
## 20:      1000 0,0,2,0,2,0,... AE004091
## 21:      1000 0,0,0,0,0,0,... BK006935
## 22:      1000 0,0,0,0,0,0,... AE016830
## 23:      1000 0,2,0,0,0,0,... AE017341
## 24:      1000 0,0,0,0,0,0,... AL009126
## 25:      1000 0,0,0,2,0,0,... AE006468
## 26:      1000 0,0,0,0,0,0,... BA000007
## 27:      1000 0,2,0,0,0,0,... AP008937
## 28:      1000 0,0,0,0,0,0,... AL591824
## 29:      1000 0,0,0,0,2,0,... CP000253
## 30:      1000 0,0,0,0,0,0,... AE004091
##     bin_width           reads  genbank
##                                                               strain
##  1:                                   Saccharomyces cerevisiae S288C
##  2:                                       Enterococcus faecalis V583
##  3:                    Cryptococcus neoformans var. neoformans JEC21
##  4:                       Bacillus subtilis subsp. subtilis str. 168
##  5: Salmonella enterica subsp. enterica serovar Typhimurium str. LT2
##  6:                              Escherichia coli O157:H7 str. Sakai
##  7:                                 Lactobacillus fermentum IFO 3956
##  8:                                     Listeria monocytogenes EGD-e
##  9:                    Staphylococcus aureus subsp. aureus NCTC 8325
## 10:                                      Pseudomonas aeruginosa PAO1
## 11:                                   Saccharomyces cerevisiae S288C
## 12:                                       Enterococcus faecalis V583
## 13:                    Cryptococcus neoformans var. neoformans JEC21
## 14:                       Bacillus subtilis subsp. subtilis str. 168
## 15: Salmonella enterica subsp. enterica serovar Typhimurium str. LT2
## 16:                              Escherichia coli O157:H7 str. Sakai
## 17:                                 Lactobacillus fermentum IFO 3956
## 18:                                     Listeria monocytogenes EGD-e
## 19:                    Staphylococcus aureus subsp. aureus NCTC 8325
## 20:                                      Pseudomonas aeruginosa PAO1
## 21:                                   Saccharomyces cerevisiae S288C
## 22:                                       Enterococcus faecalis V583
## 23:                    Cryptococcus neoformans var. neoformans JEC21
## 24:                       Bacillus subtilis subsp. subtilis str. 168
## 25: Salmonella enterica subsp. enterica serovar Typhimurium str. LT2
## 26:                              Escherichia coli O157:H7 str. Sakai
## 27:                                 Lactobacillus fermentum IFO 3956
## 28:                                     Listeria monocytogenes EGD-e
## 29:                    Staphylococcus aureus subsp. aureus NCTC 8325
## 30:                                      Pseudomonas aeruginosa PAO1
##                                                               strain
##                      species          genus             family
##  1: Saccharomyces cerevisiae  Saccharomyces Saccharomycetaceae
##  2:    Enterococcus faecalis   Enterococcus    Enterococcaceae
##  3:  Cryptococcus neoformans   Cryptococcus    Cryptococcaceae
##  4:        Bacillus subtilis       Bacillus        Bacillaceae
##  5:      Salmonella enterica     Salmonella Enterobacteriaceae
##  6:         Escherichia coli    Escherichia Enterobacteriaceae
##  7:  Lactobacillus fermentum  Lactobacillus   Lactobacillaceae
##  8:   Listeria monocytogenes       Listeria       Listeriaceae
##  9:    Staphylococcus aureus Staphylococcus  Staphylococcaceae
## 10:   Pseudomonas aeruginosa    Pseudomonas   Pseudomonadaceae
## 11: Saccharomyces cerevisiae  Saccharomyces Saccharomycetaceae
## 12:    Enterococcus faecalis   Enterococcus    Enterococcaceae
## 13:  Cryptococcus neoformans   Cryptococcus    Cryptococcaceae
## 14:        Bacillus subtilis       Bacillus        Bacillaceae
## 15:      Salmonella enterica     Salmonella Enterobacteriaceae
## 16:         Escherichia coli    Escherichia Enterobacteriaceae
## 17:  Lactobacillus fermentum  Lactobacillus   Lactobacillaceae
## 18:   Listeria monocytogenes       Listeria       Listeriaceae
## 19:    Staphylococcus aureus Staphylococcus  Staphylococcaceae
## 20:   Pseudomonas aeruginosa    Pseudomonas   Pseudomonadaceae
## 21: Saccharomyces cerevisiae  Saccharomyces Saccharomycetaceae
## 22:    Enterococcus faecalis   Enterococcus    Enterococcaceae
## 23:  Cryptococcus neoformans   Cryptococcus    Cryptococcaceae
## 24:        Bacillus subtilis       Bacillus        Bacillaceae
## 25:      Salmonella enterica     Salmonella Enterobacteriaceae
## 26:         Escherichia coli    Escherichia Enterobacteriaceae
## 27:  Lactobacillus fermentum  Lactobacillus   Lactobacillaceae
## 28:   Listeria monocytogenes       Listeria       Listeriaceae
## 29:    Staphylococcus aureus Staphylococcus  Staphylococcaceae
## 30:   Pseudomonas aeruginosa    Pseudomonas   Pseudomonadaceae
##                      species          genus             family
##                 order               class         phylum   kingdom    id
##  1: Saccharomycetales     Saccharomycetes     Ascomycota Eukaryota even1
##  2:   Lactobacillales             Bacilli     Firmicutes  Bacteria even1
##  3:       Tremellales     Tremellomycetes  Basidiomycota Eukaryota even1
##  4:        Bacillales             Bacilli     Firmicutes  Bacteria even1
##  5:  Enterobacterales Gammaproteobacteria Proteobacteria  Bacteria even1
##  6:  Enterobacterales Gammaproteobacteria Proteobacteria  Bacteria even1
##  7:   Lactobacillales             Bacilli     Firmicutes  Bacteria even1
##  8:        Bacillales             Bacilli     Firmicutes  Bacteria even1
##  9:        Bacillales             Bacilli     Firmicutes  Bacteria even1
## 10:   Pseudomonadales Gammaproteobacteria Proteobacteria  Bacteria even1
## 11: Saccharomycetales     Saccharomycetes     Ascomycota Eukaryota even2
## 12:   Lactobacillales             Bacilli     Firmicutes  Bacteria even2
## 13:       Tremellales     Tremellomycetes  Basidiomycota Eukaryota even2
## 14:        Bacillales             Bacilli     Firmicutes  Bacteria even2
## 15:  Enterobacterales Gammaproteobacteria Proteobacteria  Bacteria even2
## 16:  Enterobacterales Gammaproteobacteria Proteobacteria  Bacteria even2
## 17:   Lactobacillales             Bacilli     Firmicutes  Bacteria even2
## 18:        Bacillales             Bacilli     Firmicutes  Bacteria even2
## 19:        Bacillales             Bacilli     Firmicutes  Bacteria even2
## 20:   Pseudomonadales Gammaproteobacteria Proteobacteria  Bacteria even2
## 21: Saccharomycetales     Saccharomycetes     Ascomycota Eukaryota even3
## 22:   Lactobacillales             Bacilli     Firmicutes  Bacteria even3
## 23:       Tremellales     Tremellomycetes  Basidiomycota Eukaryota even3
## 24:        Bacillales             Bacilli     Firmicutes  Bacteria even3
## 25:  Enterobacterales Gammaproteobacteria Proteobacteria  Bacteria even3
## 26:  Enterobacterales Gammaproteobacteria Proteobacteria  Bacteria even3
## 27:   Lactobacillales             Bacilli     Firmicutes  Bacteria even3
## 28:        Bacillales             Bacilli     Firmicutes  Bacteria even3
## 29:        Bacillales             Bacilli     Firmicutes  Bacteria even3
## 30:   Pseudomonadales Gammaproteobacteria Proteobacteria  Bacteria even3
##                 order               class         phylum   kingdom    id
##       length read_length
##  1: 12073000         100
##  2:  3219000         100
##  3: 19053000         100
##  4:  4216000         100
##  5:  4858000         100
##  6:  5499000         100
##  7:  2099000         100
##  8:  2945000         100
##  9:  2822000         100
## 10:  6265000         100
## 11: 12073000         100
## 12:  3219000         100
## 13: 19053000         100
## 14:  4216000         100
## 15:  4858000         100
## 16:  5499000         100
## 17:  2099000         100
## 18:  2945000         100
## 19:  2822000         100
## 20:  6265000         100
## 21: 12073000         100
## 22:  3219000         100
## 23: 19053000         100
## 24:  4216000         100
## 25:  4858000         100
## 26:  5499000         100
## 27:  2099000         100
## 28:  2945000         100
## 29:  2822000         100
## 30:  6265000         100
##       length read_length

Where we see we actually identified the correct 10 strains. Just the coverage is really low.

hist(sl$coverage$reads[1][[1]])

Full example

We can have a look at a precomputed example from a full sample.

data(ERR260132)

head(ERR260132$abundance)
##       rank taxa_id     relative   reads        id  kingdom         phylum
## 1: species    1681 4.026444e-03   47693 ERR260132 Bacteria Actinobacteria
## 2: species   39491 4.240407e-01 5022738 ERR260132 Bacteria     Firmicutes
## 3: species  604330 9.987385e-05    1183 ERR260132 Bacteria Actinobacteria
## 4: species  166486 1.141956e-02  135264 ERR260132 Bacteria     Firmicutes
## 5: species     817 2.224578e-03   26350 ERR260132 Bacteria  Bacteroidetes
## 6: species  397865 2.158727e-04    2557 ERR260132 Bacteria  Bacteroidetes
##             class             order             family           genus
## 1: Actinobacteria Bifidobacteriales Bifidobacteriaceae Bifidobacterium
## 2:     Clostridia     Clostridiales    Lachnospiraceae   unknown_genus
## 3: Coriobacteriia  Coriobacteriales       Atopobiaceae       Olsenella
## 4:     Clostridia     Clostridiales    Lachnospiraceae       Roseburia
## 5:    Bacteroidia     Bacteroidales     Bacteroidaceae     Bacteroides
## 6:    Bacteroidia     Bacteroidales    Barnesiellaceae     Barnesiella
##                    species
## 1: Bifidobacterium bifidum
## 2:   [Eubacterium] rectale
## 3:      Olsenella umbonata
## 4:  Roseburia intestinalis
## 5:    Bacteroides fragilis
## 6: Barnesiella viscericola

This gives us better coverage:

co <- ERR260132$coverage
co[, mean_coverage := sum(reads[[1]]) / length, by = "genbank"]

plot(co[order(-mean_coverage)][1, reads[[1]]], type = "l")

Where we see that the majority of that genome has not been mapped.