Introduction

palmid is a containerized analysis suite and R-package for the classification of viral RNA-dependent RNA Polymerases (RdRP) based on the palmprint sub-domain and the RNA viral palmprint database palmdb.

RdRP Palmprint
=============================================
The `palmprint` is an ~100 aa segment of RdRP
encompassing three conserved catalytic motifs
"A", "B", and "C" within the palm sub-domain.

Web Version

palmID is available as a free web-app at https://serratus.io/palmid

Local Install

palmid (container)

# Download the `palmid` container
sudo docker pull serratusbio/palmid:latest
# Alternative: build container locally

# Clone repository
git clone https://github.com/ababaian/palmid.git && cd palmid

# Requires `docker` (>= v20.10)
sudo docker build -t serratusbio/palmid:latest ./

palmid (R package Only)

# R (>= v4.0.3)
# Install dependencies
install.packages("devtools")
devtools::install_github("ababaian/palmid")

# Load libraries
library("palmid")

# Install Mapping Functions for static maps (optional)
#  'libudunits2-dev' and geo system libraries needed
#   sudo apt-get install -y  libudunits2-dev \
#                            libgdal-dev     \ 
#                            libgeos-dev     \
#                            libproj-dev     \
install.packages("sf")
install.packages("rnaturalearth")

Local usage

0) Input

Input a .fa sequence file containing an RdRP. Here we show a ‘microassembly’ open-reading-frame from a sequencing library of Waxsystermes termites (SRR9968562) as derived from the Serratus: Finding Novel Viruses Tutorial.

data/waxsys.fa

>SRR9968562_waxsystermes_virus_microassembly
PIWDRVLEPLMRASPGIGRYMLTDVSPVGLLRVFKEKVDTTPHMPPEGMEDFKKASKEVE
KTLPTTLRELSWDEVKEMIRNDAAVGDPRWKTALEAKESEEFWREVQAEDLNHRNGVCLR
GVFHTMAKREKKEKNKWGQKTSRMIAYYDLIERACEMRTLGALNADHWAGEENTPEGVSG
IPQHLYGEKALNRLKMNRMTGETTEGQVFQGDIAGWDTRVSEYELQNEQRICEERAESED
HRRKIRTIYECYRSPIIRVQDADGNLMWLHGRGQRMSGTIVTYAMNTITNAIIQQAVSKD
LGNTYGRENRLISGDDCLVLYDTQHPEETLVAAFAKYGKVLKFEPGEPTWSKNIENTWFC
SHTYSRVKVGNDIRIMLDRSEIEILGKARIVLGGYKTGEVEQAMAKGYANYLLLTFPQRR
NVRLAANMVRAIVPRGLLPMGRAKDPWWREQPWMSTNNMIQAFNQIWEGWPPISSMKDIK
YVGRAREQMLDST

Run the containerized palmid workflow

# Run palmid analysis suite
# uses the "scripts/palmid.sh" script as entrypoint
#
# palmid -i <input_fasta> -o <output_path>
# -v | -w flags are to mount the work dir into the conntainer
#
sudo docker run  -v `pwd`:`pwd` -w `pwd`  \
  --entrypoint "/bin/bash" serratusbio/palmid:latest \
  /home/palmid/palmid.sh -i data/waxsys.fa -d test -o waxsys

1) Palmprint Report

palmscan will analyze the RdRP and a .txt report shows the catalytic motifs and their scores. It will also report the amino acid sequence “trimmed” to its palmprint sub-sequence.

data/waxsys.txt

>SRR9968562_waxsystermes_virus_microassembly
   A:209-220(11.8)      B:277-290(19.3)      C:312-319(14.3)
   FQGDIAGWDTRV    <56> SGTIVTYAMNTITN  <21> ISGDDCLV  [111]
   |  |.+.||++|         ||  .||. |||||       .|||||||
   lenDyskFDksq         SGdanTslGNTltn       vsGDDsvv
Score 55.4, high-confidence-RdRP: high-PSSM-score.reward-DDGGDD.good-segment-length.

data/waxsys.trim.fa

>SRR9968562_waxsystermes_virus_microassembly
FQGDIAGWDTRVSEYELQNEQRICEERAESEDHRRKIRTIYECYRSPIIRVQDADGNLMW
LHGRGQRMSGTIVTYAMNTITNAIIQQAVSKDLGNTYGRENRLISGDDCLV

The palmid R package visualizes this data, showing the relative palmprint scores and length-distributions for the input sequence vs a control set 15,000 GenBank RdRP palmprints in palmdb.

data/waxsys_pp.png

Waxsystermes virus palmprint report

2) Comparison to PalmDB

Input RdRP palmprint is aligned against palmdb using diamond to retrieve similar viruses. The data/waxsys.pro alignment file is visualized in the palmid R package to show the relative similarity of RdRP palmprints.

data/waxsys_pro.png

Waxsystermes virus palmdb report

Known virus taxonomy is extracted from palmdb-matches (when available) and the species/family/phylum are shown as a function of percent-identity to the input sequence.

data/waxsys_tax.png

Waxsystermes virus palmdb-tax report

A multiple sequence alignment of the top 10 palmprint hits is produced for manual validation. A central observation here is that the A,B,C catalytic motifs align to one another.

data/waxsys.msa.fa (top 10 hits)

>u18590_41.8
FADDTAGWDTRITVADLENEAKILDRMDG--DHKRLARAIVELTYRHKVVKVMRPSSSG-GTVMDVISREDQRGSGQVVTYALNTFTNLAVQLIRCMEGEGLIGPEDVEDLRKGKLPTIKNWLLKNGTERLSRMAVSGDDCVV
>u8640_41.4
YADDTAGWDTRITECDLRNEAHIMEYMEN--EHRKLARAIFELTYKHKVVKVMRP-GKG-VPLMDIISREDQRGSGQVVTYALNTFTNLVVQLIRMAEAECVLTPEDLHEMSQSAKLRLLKWLKEEGWERLTRMAVSGDDCVV
>u181012_43.0
CSSDIAGFDTKVSMYTLQLEYMFCCLLGITSVT---AKNLYRI-YAHPHILV--PQVSE-YARVELLQGRGQRMSGTQVTYPMNTITRMALTILQLYTSKRQ----TLT-PDQFVLHYMKCRL------KA-RSCISGDDEVL
>u32314_41.9
CADDIAGWDTRIGVIMQSMECRFICALTKSKNLRKKIRAMYRL-YAYPHMLI--PRHTDRFVRSELVRGRGSVMSGRIVTYSMNTISRIAVSLLQQAVADKV----EIKDLREYARMEMSGLTLDGKPSRW-GGCTSGDDSFR
>u253902_41.8
CSSDIAGFDTRVSLRRLSDEARFHSILGAPDIC----HMFYRI-YAYPHILV--PTLDG---KTELLKGRGQRMSGTGPTYSMNTITRIVLMFLQIMVSVGV----DVSDPEN-VERAFHTIM---ADKRW-QGGVSGDDEFV
>u38234_41.7
VSDDIAGFDTRVSLTTLSLENMFVKMLGGNLTH----EHMYRL-YGYPMIIV--PIDSE-YNRSELLRGRGQRMSGSNPTYSMNTITRIAVGLLQLSVVMKI----DEDDILLWVEKQMNKKT------SDMTGCVSGDDATF
>u32970_41.1
VSDDIAGFDTRIGLYFLSLENHFIRMLGGGEIH----TLMYRL-YAYPHILI--PMASE-FVRSQLLKGRGQRMSGTNVTYSMNTITRICVCLLQYAIAKDI----PLNELHDWTMQMMKQNS------PL-QGVVSGDDASF
>u5157_44.7
IQDDTAGWDTRLHDDVLECEQSFLCDFAESEEHIKHILRIYKN-YRNPMIKL--TDDSG--TRDLILIGKGQRCSGTVVTYSMNTITNTVVQMMRMQEVLEL-----------SNEECLHKMM------------VSGDDCLL
>SRR9968562_waxsystermes_virus_microassembly
FQGDIAGWDTRVSEYELQNEQRICEERAESEDHRRKIRTIYEC-YRSPIIRV--QDADG---NLMWLHGRGQRMSGTIVTYAMNTITN---AIIQQAVSKDL-----------GNTYGRENRL------------ISGDDCLV
>u128522_100.0
FQGDIAGWDTRVSEYELQNEQRICEERAESEDHRRKIRTIYEC-YRSPIIRV--QDADG---NLMWLHGRGQRMSGTIVTYAMNTITN---AIIQQAVSKDL-----------GNTYGRENRL------------ISGDDCLV
>u18016_61.3
FQGDISGWDTRVSEYELEWEQRTLVERAQTEGHKRAIMTQYEC-YRNPIIKM--PQQGG---REVWLSGRGQRMSGTNVTYYCNTLTN---AVLQEAVFTDL---------FGISEVARKRRM------------ISGDDCCC

3) Cross-analysis to SRA metadata

The palmid.Rmd notebook performs an analysis of the detection/alignment files produced above. Palmprints matching the input-sequence are cross-referenced against all processed SRA sequencing libraries. Geo-spatial data (when available) and timeline of the matching sequencing runs are reported. A full example of the output is available here

data/waxsys_geo.png

Waxsystermes virus geospatial

The organism reported with each sequencing run is conglomerated into a wordcloud to visualize possible hosts. Current default will report the organisms associated with all palmprint-matches, for specificity to the input virus species use a threshold of 90%.

data/waxsys_orgn.png

Waxsystermes virus organism

References

A. Babaian and R. C. Edgar (2021), Ribovirus classification by a polymerase barcode sequence, biorxiv https://doi.org/10.1101/2021.03.02.433648

R. C. Edgar et al. (2021), Petabase-scale sequence alignment catalyses viral discovery, biorxiv https://www.biorxiv.org/content/10.1101/2020.08.07.241729v2