Marker data
Either clone the THAPBI PICT source code repository, or decompress the
latest source code release (.tar.gz
file). You should find it contains
a directory examples/woody_hosts/
which is for this example.
Shell scripts setup.sh
and run.sh
should reproduce the analysis
discussed.
The documentation goes through running each step of the analysis gradually,
before finally calling pipeline command to do it all together. We provide
script run.sh
to do the final run-though automatically (first without
any metadata, then again with it), but encourage you to follow along the
individual steps first.
FASTQ data
The raw data is from two Illumina MiSeq runs, a whole 96-well plate from 2016, and about half the samples from a second 96-well plate sequenced in 2017 (where the rest of the plate was samples from a separate ITS1 study). There are multiple replicates from each of 14 sample sites, plus controls. The raw FASTQ files are too large to include with the THAPBI PICT source code.
Script setup.sh
will download the raw FASTQ files for Riddell et al.
(2019) from https://doi.org/10.5281/zenodo.3342957
It will download 244 raw FASTQ files (122 pairs), about 215MB on disk
Amplicon primers & reference sequences
The ITS1 primers used here match the THAPBI PICT defaults, so the default database can also be used.
Metadata
The provided file metadata.tsv
is an expanded version of Supplementary
Table 1 from the original paper, adding a column for the Illumina MiSeq sample
names, rows for the controls.
The 16 columns are as follows, where 4 to 15 are in pairs for tree/shrub broad taxonomic grouping and health status (H, healthy; D, symptoms/stump/dead):
Anonymised site number (with leading zero, “01” to “14”), or control name
Approximate altitude at centre
Underlying soil type
Healthy Cupressaceae
Diseased Cupressaceae
Healthy other conifers
Diseased other conifers
Healthy Ericaceae
Diseased Ericaceae
Healthy Fagaceae or Nothofagaceae
Diseased Fagaceae or Nothofagaceae
Healthy other angiosperms
Diseased other angiosperms
Healthy other
Diseased other
MiSeq Sample(s) (semi-colon separated list)
When calling THAPBI PICT, the meta data commands are given as follows:
$ thapbi_pict ... -t metadata.tsv -x 16 -c 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15
These settings are described in detail later (see Metadata). This example is important in that column 16 contains multiple entries where a site had multiple sequenced samples (replicates).
Other files
Subdirectory expected/
contains four plain text tab-separated files,
describing the expected species in some mock community positive controls:
DNA15MIX.known.tsv
DNA10MIX_bycopynumber.known.tsv
DNA10MIX_diluted25x.known.tsv
DNA10MIX_undiluted.known.tsv