# Inference and Analysis of Synteny Networks

Fabricio Almeida-Silva1,2, Tao Zhao3, Kristian K Ullrich4 and Yves Van de Peer1,2,5,6

1VIB-UGent Center for Plant Systems Biology, Ghent, Belgium
2Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium
3State Key Laboratory of Crop Stress Biology for Arid Areas/Shaanxi Key Laboratory of Apple, College of Horticulture, Northwest A&F University, Yangling, China
4Department of Evolutionary Biology, Max Planck Institute For Evolutionary Biology, Ploen, Germany
5College of Horticulture, Academy for Advanced Interdisciplinary Studies, Nanjing Agricultural University, Nanjing, China
6Center for Microbial Ecology and Genomics, Department of Biochemistry, Genetics and Microbiology, University of Pretoria, Pretoria, South Africa

#### 24 July 2025

# 1 Introduction

The analysis of synteny (i.e., conserved gene content and order in a genomic segment across species) can help understand the trajectory of duplicated genes through evolution. In particular, synteny analyses are widely used to investigate the evolution of genes derived from whole-genome duplication (WGD) events, as they can reveal genomic rearrangements that happened following the duplication of all chromosomes. However, synteny analysis are typically performed in a pairwise manner, which can be difficult to explore and interpret when comparing several species. To understand global patterns of synteny, Zhao and Schranz (2017) proposed a network-based approach to analyze synteny. In synteny networks, genes in a given syntenic block are represented as nodes connected by an edge. Synteny networks have been used to explore, among others, global synteny patterns in mammalian and angiosperm genomes (Zhao and Schranz 2019), the evolution of MADS-box transcription factors (Zhao et al. 2017), and infer a microsynteny-based phylogeny for angiosperms (Zhao et al. 2021). syntenet is a package that can be used to infer synteny networks from protein sequences and perform downstream network analyses that include:

- Network clustering using the Infomap algorithm;

- Phylogenomic profiling, which consists in identifying which species contain which clusters. This analysis can reveal highly conserved synteny clusters and taxon-specific ones (e.g., family- and order-specific clusters);

- Microsynteny-based phylogeny reconstruction with maximum likelihood, which can be achieved by inferring a phylogeny from a binary matrix of phylogenomic profiles with IQ-TREE.

# 2 Installation

syntenet can be installed from Bioconductor with the following code:

    if(!requireNamespace('BiocManager', quietly = TRUE))
      install.packages('BiocManager')

    BiocManager::install("syntenet")

    # Load package after installation
    library(syntenet)

# 3 Data description

For this vignette, we will use the proteomes and gene annotation of the algae species Ostreococcus lucimarinus and Ostreococcus sp RCC809, which were obtained from Pico-PLAZA 3.0 (Vandepoele et al. 2013).

    # Protein sequences
    data(proteomes)
    head(proteomes)
    #> $Olucimarinus
    #> AAStringSet object of length 1901:
    #>        width seq                                            names
    #>    [1]   911 MTTMADERASIARVSVVKYGAI...VQLYTYPGSTNDPNFLLKLA* OL01G00010
    #>    [2]   789 MGGRRCFCSRSSPVGVGAPAPA...PPQCGADIEAGSEPPPDKCG* OL01G00020
    #>    [3]   618 MTRAKDAIVVDDGNDDDDDDDD...RDASASLALALAFSSEESVV* OL01G00030
    #>    [4]   547 MPTKAQCWVVSYARVRDGASRS...TGSVSARASIFGEQASFRKA* OL01G00040
    #>    [5]   319 MFTASHTTSKVTLRARVATQPR...HNGMALWRETTPKDSLIPAL* OL01G00050
    #>    ...   ... ...
    #> [1897]   106 MAANDGETKLPEDGWIQPCFAC...RAIVDQVGGEHLKGSLMPIE* OL03G05910
    #> [1898]    70 RMGIVKLATDGSVWVHSPIELD...QQWKDAYPGATLYACPGLKSK OL03G05920
    #> [1899]   680 MDDAHDARWATTSARDGERARA...RSVGPSASDKILEALFPVAD* OL03G05930
    #> [1900]   179 MRAVRERSKANLAARVKEEATR...ELERTRELFARARVRAYECI* OL03G05940
    #> [1901]    83 MFVREARRAIPRFIKDPPQAFH...ESGDVRSVEGEVCGAVLVDE* OL03G05950
    #>
    #> $OspRCC809
    #> AAStringSet object of length 1433:
    #>        width seq                                            names
    #>    [1]   274 MASTTGSAARRVFVDVEKTVNG...DVLSLGQGSLSGESSSSDEE* ORCC809_01G00010
    #>    [2]   175 MDQMRAANAQRSYLLFFVLFFL...SRRLLGRLDSEHTDLHPSWR* ORCC809_01G00020
    #>    [3]   403 MTAPRVRASRRATATAAATVTA...LTERDLRYMEPKATIEEWMG* ORCC809_01G00030
    #>    [4]   217 MTIDADGDDTLAPHAPAHGEVS...LIRLRGVEKTPTVDPPPPPP* ORCC809_01G00040
    #>    [5]  1691 RIEADEKSLLVFGKESPVRTAC...VRMGNNVVTSRYASSESEEDV ORCC809_01G00050
    #>    ...   ... ...
    #> [1429]   428 MVDANATTQTFVLEAEQELRVE...DLPSNVLLVGNLKWLGEDGK* ORCC809_03G02980
    #> [1430]   378 MSVPRTTLRRIPLGNARDVLVT...ETLKAIDAVHAQCRDPCIAT* ORCC809_03G02990
    #> [1431]  1156 MRATSAPSIVSFVARVACLFVA...CAFGTSLASFVVERARRLEN* ORCC809_03G03000
    #> [1432]   541 MAITVFLTDHGRRASALTFLVV...GFGVGAVKFMLAPEMVKSLA* ORCC809_03G03010
    #> [1433]   289 MSLSSLRSFSRSISSAPGGRSC...EPEEPEPEEPEPEEPEEPEP* ORCC809_03G03020

    # Annotation (ranges)
    data(annotation)
    head(annotation)
    #> GRangesList object of length 2:
    #> $Olucimarinus
    #> GRanges object with 1903 ranges and 4 metadata columns:
    #>          seqnames        ranges strand |     type          ID        Name
    #>             <Rle>     <IRanges>  <Rle> | <factor> <character> <character>
    #>      [1]    Chr_1      939-3671      - |     gene  OL01G00010  OL01G00010
    #>      [2]    Chr_1     3907-6927      + |     gene  OL01G00020  OL01G00020
    #>      [3]    Chr_1     7085-9160      + |     gene  OL01G00030  OL01G00030
    #>      [4]    Chr_1    9830-11480      + |     gene  OL01G00040  OL01G00040
    #>      [5]    Chr_1   11467-12599      - |     gene  OL01G00050  OL01G00050
    #>      ...      ...           ...    ... .      ...         ...         ...
    #>   [1899]    Chr_3 977435-977752      - |     gene  OL03G05910  OL03G05910
    #>   [1900]    Chr_3 978702-978911      - |     gene  OL03G05920  OL03G05920
    #>   [1901]    Chr_3 979281-981320      - |     gene  OL03G05930  OL03G05930
    #>   [1902]    Chr_3 981778-982314      + |     gene  OL03G05940  OL03G05940
    #>   [1903]    Chr_3 982498-982746      + |     gene  OL03G05950  OL03G05950
    #>              gene_id
    #>          <character>
    #>      [1]  OL01G00010
    #>      [2]  OL01G00020
    #>      [3]  OL01G00030
    #>      [4]  OL01G00040
    #>      [5]  OL01G00050
    #>      ...         ...
    #>   [1899]  OL03G05910
    #>   [1900]  OL03G05920
    #>   [1901]  OL03G05930
    #>   [1902]  OL03G05940
    #>   [1903]  OL03G05950
    #>   -------
    #>   seqinfo: 6 sequences from an unspecified genome; no seqlengths
    #>
    #> $OspRCC809
    #> GRanges object with 1433 ranges and 4 metadata columns:
    #>          seqnames        ranges strand |     type               ID
    #>             <Rle>     <IRanges>  <Rle> | <factor>      <character>
    #>      [1]    chr_1      321-1142      - |     gene ORCC809_01G00010
    #>      [2]    chr_1     1463-2089      + |     gene ORCC809_01G00020
    #>      [3]    chr_1     2162-3370      - |     gene ORCC809_01G00030
    #>      [4]    chr_1     3774-4424      - |     gene ORCC809_01G00040
    #>      [5]    chr_1     4693-9924      - |     gene ORCC809_01G00050
    #>      ...      ...           ...    ... .      ...              ...
    #>   [1429]    chr_3 504915-506198      - |     gene ORCC809_03G02980
    #>   [1430]    chr_3 506377-507510      + |     gene ORCC809_03G02990
    #>   [1431]    chr_3 507856-511323      + |     gene ORCC809_03G03000
    #>   [1432]    chr_3 511533-513155      - |     gene ORCC809_03G03010
    #>   [1433]    chr_3 513841-514707      + |     gene ORCC809_03G03020
    #>                      Name          gene_id
    #>               <character>      <character>
    #>      [1] ORCC809_01G00010 ORCC809_01G00010
    #>      [2] ORCC809_01G00020 ORCC809_01G00020
    #>      [3] ORCC809_01G00030 ORCC809_01G00030
    #>      [4] ORCC809_01G00040 ORCC809_01G00040
    #>      [5] ORCC809_01G00050 ORCC809_01G00050
    #>      ...              ...              ...
    #>   [1429] ORCC809_03G02980 ORCC809_03G02980
    #>   [1430] ORCC809_03G02990 ORCC809_03G02990
    #>   [1431] ORCC809_03G03000 ORCC809_03G03000
    #>   [1432] ORCC809_03G03010 ORCC809_03G03010
    #>   [1433] ORCC809_03G03020 ORCC809_03G03020
    #>   -------
    #>   seqinfo: 6 sequences from an unspecified genome; no seqlengths

# 4 Importing data to the R session

To detect synteny and infer synteny networks, syntenet requires two objects as input:

- seq: A list of `AAStringSet` objects containing the translated sequences of primary transcripts for each species.
- annotation: A `GRangesList` or `CompressedGRangesList` object containing the coordinates for the genes in seq.

If you have whole-genome protein sequences in FASTA files, store all FASTA files in the same directory and use the function `fasta2AAStringSetlist()` to read all FASTA files into a list of `AAStringSet` objects.

Likewise, if you have gene annotation in GFF/GFF3/GTF files, store all files in the same directory and use the function `gff2GRangesList()` to read all GFF/GFF3/GTF files into a `GRangesList object`.

For a demonstration, we will read example FASTA and GFF3 files stored in subdirectories named sequences/ and annotation/, which are located in the `extdata/` directory of this package.

## 4.1 From FASTA files to a list of `AAStringSet` objects

Here is how you can use `fasta2AAStringSetlist()` to read FASTA files in a directory as a list of `AAStringSet` objects.

    # Path to directory containing FASTA files
    fasta_dir <- system.file("extdata", "sequences", package = "syntenet")
    fasta_dir
    #> [1] "E:/biocbuild/bbs-3.21-bioc/tmpdir/RtmpCSOpbE/Rinst11cfc4987523/syntenet/extdata/sequences"

    dir(fasta_dir) # see the contents of the directory
    #> [1] "Olucimarinus.fa.gz" "OspRCC809.fa.gz"

    # Read all FASTA files in `fasta_dir`
    aastringsetlist <- fasta2AAStringSetlist(fasta_dir)
    aastringsetlist
    #> $Olucimarinus
    #> AAStringSet object of length 100:
    #>       width seq                                             names
    #>   [1]   911 MTTMADERASIARVSVVKYGAI...DVQLYTYPGSTNDPNFLLKLA* OL01G00010
    #>   [2]   789 MGGRRCFCSRSSPVGVGAPAPA...FPPQCGADIEAGSEPPPDKCG* OL01G00020
    #>   [3]   618 MTRAKDAIVVDDGNDDDDDDDD...DRDASASLALALAFSSEESVV* OL01G00030
    #>   [4]   547 MPTKAQCWVVSYARVRDGASRS...VTGSVSARASIFGEQASFRKA* OL01G00040
    #>   [5]   319 MFTASHTTSKVTLRARVATQPR...LHNGMALWRETTPKDSLIPAL* OL01G00050
    #>   ...   ... ...
    #>  [96]   476 MVPARNFLDGANAREVELDRVV...VMRKLREPDSVARLAGQTGVR* OL01G00960
    #>  [97]   771 MARHRGTRGGWNATTTEGGDGR...SIPDDGFDESSSVSASTIDGF* OL01G00970
    #>  [98]   494 MDSEFWGCVIPAGRAVRVEVAT...FIKSRKDLFTIDGAYVRLVKK* OL01G00980
    #>  [99]   264 VRAIVGATTRIQTRAPPRANHR...DWSFISDEFQDDASDSEVIDR* OL01G00990
    #> [100]   565 MQLDAFRKATVKGVATRVGGAD...QLADLLRKNMGVPAKFIDAQN* OL01G01000
    #>
    #> $OspRCC809
    #> AAStringSet object of length 100:
    #>       width seq                                             names
    #>   [1]   274 MASTTGSAARRVFVDVEKTVNG...WDVLSLGQGSLSGESSSSDEE* ORCC809_01G00010
    #>   [2]   175 MDQMRAANAQRSYLLFFVLFFL...SSRRLLGRLDSEHTDLHPSWR* ORCC809_01G00020
    #>   [3]   403 MTAPRVRASRRATATAAATVTA...ALTERDLRYMEPKATIEEWMG* ORCC809_01G00030
    #>   [4]   217 MTIDADGDDTLAPHAPAHGEVS...SLIRLRGVEKTPTVDPPPPPP* ORCC809_01G00040
    #>   [5]  1691 RIEADEKSLLVFGKESPVRTAC...SVRMGNNVVTSRYASSESEEDV ORCC809_01G00050
    #>   ...   ... ...
    #>  [96]   357 MSRGLADNWDDAEGYYCARIGE...TVNEALQHPFIVERIRTTAPN* ORCC809_01G00960
    #>  [97]   164 MAMDSFRSAPRSRRRVEATSRE...SKPVKPVREPVRMVEASTGAH* ORCC809_01G00970
    #>  [98]    85 MPEGTVFIGNIPYDATESSLTE...NLNAREYNGRQLRVDHAETMKG ORCC809_01G00980
    #>  [99]   229 MKGGGGASGAAASANGNGAVGG...PDQRAQVEYLRQLAAQQGMVR* ORCC809_01G00990
    #> [100]   103 RKAGGERWEDSSLAEWPENDFR...EMAGKFIGNRPVKLRKSAWNER ORCC809_01G01000

And that’s it! Now you have a list of `AAStringSet` objects.

## 4.2 From GFF/GTF files to a `GRangesList` object

Here is how you can use `gff2GRangesList()` to read GFF/GFF3/GTF files in a directory as a `GRangesList` object.

    # Path to directory containing FASTA files
    gff_dir <- system.file("extdata", "annotation", package = "syntenet")
    gff_dir
    #> [1] "E:/biocbuild/bbs-3.21-bioc/tmpdir/RtmpCSOpbE/Rinst11cfc4987523/syntenet/extdata/annotation"

    dir(gff_dir) # see the contents of the directory
    #> [1] "Olucimarinus.gff3.gz" "OspRCC809.gff3.gz"

    # Read all FASTA files in `fasta_dir`
    grangeslist <- gff2GRangesList(gff_dir)
    grangeslist
    #> GRangesList object of length 2:
    #> $Olucimarinus
    #> GRanges object with 100 ranges and 7 metadata columns:
    #>         seqnames        ranges strand |      source     type     score
    #>            <Rle>     <IRanges>  <Rle> |    <factor> <factor> <numeric>
    #>     [1]    Chr_1      939-3671      - | rtracklayer     gene        NA
    #>     [2]    Chr_1     3907-6927      + | rtracklayer     gene        NA
    #>     [3]    Chr_1     7085-9160      + | rtracklayer     gene        NA
    #>     [4]    Chr_1    9830-11480      + | rtracklayer     gene        NA
    #>     [5]    Chr_1   11467-12599      - | rtracklayer     gene        NA
    #>     ...      ...           ...    ... .         ...      ...       ...
    #>    [96]    Chr_1 170975-172402      + | rtracklayer     gene        NA
    #>    [97]    Chr_1 172445-174757      - | rtracklayer     gene        NA
    #>    [98]    Chr_1 175358-176839      + | rtracklayer     gene        NA
    #>    [99]    Chr_1 176901-177692      - | rtracklayer     gene        NA
    #>   [100]    Chr_1 177742-179436      - | rtracklayer     gene        NA
    #>             phase          ID        Name     gene_id
    #>         <integer> <character> <character> <character>
    #>     [1]      <NA>  OL01G00010  OL01G00010  OL01G00010
    #>     [2]      <NA>  OL01G00020  OL01G00020  OL01G00020
    #>     [3]      <NA>  OL01G00030  OL01G00030  OL01G00030
    #>     [4]      <NA>  OL01G00040  OL01G00040  OL01G00040
    #>     [5]      <NA>  OL01G00050  OL01G00050  OL01G00050
    #>     ...       ...         ...         ...         ...
    #>    [96]      <NA>  OL01G00960  OL01G00960  OL01G00960
    #>    [97]      <NA>  OL01G00970  OL01G00970  OL01G00970
    #>    [98]      <NA>  OL01G00980  OL01G00980  OL01G00980
    #>    [99]      <NA>  OL01G00990  OL01G00990  OL01G00990
    #>   [100]      <NA>  OL01G01000  OL01G01000  OL01G01000
    #>   -------
    #>   seqinfo: 2 sequences from an unspecified genome; no seqlengths
    #>
    #> $OspRCC809
    #> GRanges object with 100 ranges and 7 metadata columns:
    #>         seqnames        ranges strand |      source     type     score
    #>            <Rle>     <IRanges>  <Rle> |    <factor> <factor> <numeric>
    #>     [1]    chr_1      321-1142      - | rtracklayer     gene        NA
    #>     [2]    chr_1     1463-2089      + | rtracklayer     gene        NA
    #>     [3]    chr_1     2162-3370      - | rtracklayer     gene        NA
    #>     [4]    chr_1     3774-4424      - | rtracklayer     gene        NA
    #>     [5]    chr_1     4693-9924      - | rtracklayer     gene        NA
    #>     ...      ...           ...    ... .         ...      ...       ...
    #>    [96]    chr_1 165459-166529      - | rtracklayer     gene        NA
    #>    [97]    chr_1 166654-167213      - | rtracklayer     gene        NA
    #>    [98]    chr_1 167296-167550      + | rtracklayer     gene        NA
    #>    [99]    chr_1 167542-168228      + | rtracklayer     gene        NA
    #>   [100]    chr_1 168639-168947      - | rtracklayer     gene        NA
    #>             phase               ID             Name          gene_id
    #>         <integer>      <character>      <character>      <character>
    #>     [1]      <NA> ORCC809_01G00010 ORCC809_01G00010 ORCC809_01G00010
    #>     [2]      <NA> ORCC809_01G00020 ORCC809_01G00020 ORCC809_01G00020
    #>     [3]      <NA> ORCC809_01G00030 ORCC809_01G00030 ORCC809_01G00030
    #>     [4]      <NA> ORCC809_01G00040 ORCC809_01G00040 ORCC809_01G00040
    #>     [5]      <NA> ORCC809_01G00050 ORCC809_01G00050 ORCC809_01G00050
    #>     ...       ...              ...              ...              ...
    #>    [96]      <NA> ORCC809_01G00960 ORCC809_01G00960 ORCC809_01G00960
    #>    [97]      <NA> ORCC809_01G00970 ORCC809_01G00970 ORCC809_01G00970
    #>    [98]      <NA> ORCC809_01G00980 ORCC809_01G00980 ORCC809_01G00980
    #>    [99]      <NA> ORCC809_01G00990 ORCC809_01G00990 ORCC809_01G00990
    #>   [100]      <NA> ORCC809_01G01000 ORCC809_01G01000 ORCC809_01G01000
    #>   -------
    #>   seqinfo: 2 sequences from an unspecified genome; no seqlengths

And now you have a `GRangesList` object.

# 5 Data preprocessing

The first part of the pipeline consists in processing the data to make it match a standard structure. However, before processing the data for synteny detection, you must use the function `check_input()` to check if your data can enter the pipeline. This function checks the input data for 3 required conditions:

1. Names of seq list (i.e., `names(seq)`) match the names of annotation `GRangesList`/`CompressedGRangesList` (i.e., `names(annotation)`)

2. For each species (list elements), the number of sequences in seq is not greater than the number of genes in annotation. This is a way to ensure users do not input the translated sequences for multiple isoforms of the same gene (generated by alternative splicing). Ideally, the number of sequences in seq should be equal to the number of genes in annotation, but this may not always stand true because of non-protein-coding genes.

3. For each species, sequence names (i.e., `names(seq[[x]])`, equivalent to FASTA headers) match gene names in `annotation`. By default, syntenet looks for gene IDs in a column named “gene_id” in the GRanges objects (default field in GFF3 files). If your gene IDs are in a different column (e.g., “Name”), you can specify it in the gene_field parameter of `check_input()` and `process_input()`.

Let’s check if the example data sets satisfy these 3 criteria:

    check_input(proteomes, annotation)
    #> [1] TRUE

As you can see, the data passed the checks. Now, let’s process them with the function `process_input()`.

This function processes the input sequences and annotation to:

1. Remove whitespace and anything after it in sequence names (i.e., `names(seq[[x]])`, which is equivalent to FASTA headers), if there is any.

2. Add a unique species identifier to sequence names. The species identifier consists of the first 3-5 strings of the element name. For instance, if the first element of the seq list is named “Athaliana”, each sequence in it will have an identifier “Atha_” added to the beginning of each gene name (e.g., Atha_AT1G01010).

3. If sequences have an asterisk (*) representing stop codon, remove it.

4. Add a unique species identifier (same as above) to gene and chromosome names of each element of the annotation `GRangesList`/`CompressedGRangesList`.

5. Filter each element of the annotation `GRangesList`/`CompressedGRangesList` to keep only seqnames, ranges, and gene ID.

Let’s process our input data:

    pdata <- process_input(proteomes, annotation)

    # Looking at the processed data
    pdata$seq
    #> $Olucimarinus
    #> AAStringSet object of length 1901:
    #>        width seq                                            names
    #>    [1]   910 MTTMADERASIARVSVVKYGAI...DVQLYTYPGSTNDPNFLLKLA Olu_OL01G00010
    #>    [2]   788 MGGRRCFCSRSSPVGVGAPAPA...FPPQCGADIEAGSEPPPDKCG Olu_OL01G00020
    #>    [3]   617 MTRAKDAIVVDDGNDDDDDDDD...DRDASASLALALAFSSEESVV Olu_OL01G00030
    #>    [4]   546 MPTKAQCWVVSYARVRDGASRS...VTGSVSARASIFGEQASFRKA Olu_OL01G00040
    #>    [5]   318 MFTASHTTSKVTLRARVATQPR...LHNGMALWRETTPKDSLIPAL Olu_OL01G00050
    #>    ...   ... ...
    #> [1897]   105 MAANDGETKLPEDGWIQPCFAC...LRAIVDQVGGEHLKGSLMPIE Olu_OL03G05910
    #> [1898]    69 RMGIVKLATDGSVWVHSPIELD...AQQWKDAYPGATLYACPGLKS Olu_OL03G05920
    #> [1899]   679 MDDAHDARWATTSARDGERARA...ARSVGPSASDKILEALFPVAD Olu_OL03G05930
    #> [1900]   178 MRAVRERSKANLAARVKEEATR...LELERTRELFARARVRAYECI Olu_OL03G05940
    #> [1901]    82 MFVREARRAIPRFIKDPPQAFH...QESGDVRSVEGEVCGAVLVDE Olu_OL03G05950
    #>
    #> $OspRCC809
    #> AAStringSet object of length 1433:
    #>        width seq                                            names
    #>    [1]   273 MASTTGSAARRVFVDVEKTVNG...WDVLSLGQGSLSGESSSSDEE Osp_ORCC809_01G00010
    #>    [2]   174 MDQMRAANAQRSYLLFFVLFFL...SSRRLLGRLDSEHTDLHPSWR Osp_ORCC809_01G00020
    #>    [3]   402 MTAPRVRASRRATATAAATVTA...ALTERDLRYMEPKATIEEWMG Osp_ORCC809_01G00030
    #>    [4]   216 MTIDADGDDTLAPHAPAHGEVS...SLIRLRGVEKTPTVDPPPPPP Osp_ORCC809_01G00040
    #>    [5]  1690 RIEADEKSLLVFGKESPVRTAC...SVRMGNNVVTSRYASSESEED Osp_ORCC809_01G00050
    #>    ...   ... ...
    #> [1429]   427 MVDANATTQTFVLEAEQELRVE...GDLPSNVLLVGNLKWLGEDGK Osp_ORCC809_03G02980
    #> [1430]   377 MSVPRTTLRRIPLGNARDVLVT...KETLKAIDAVHAQCRDPCIAT Osp_ORCC809_03G02990
    #> [1431]  1155 MRATSAPSIVSFVARVACLFVA...ACAFGTSLASFVVERARRLEN Osp_ORCC809_03G03000
    #> [1432]   540 MAITVFLTDHGRRASALTFLVV...PGFGVGAVKFMLAPEMVKSLA Osp_ORCC809_03G03010
    #> [1433]   288 MSLSSLRSFSRSISSAPGGRSC...EEPEEPEPEEPEPEEPEEPEP Osp_ORCC809_03G03020
    pdata$annotation
    #> $Olucimarinus
    #> GRanges object with 1903 ranges and 1 metadata column:
    #>           seqnames        ranges strand |           gene
    #>              <Rle>     <IRanges>  <Rle> |    <character>
    #>      [1] Olu_Chr_1      939-3671      - | Olu_OL01G00010
    #>      [2] Olu_Chr_1     3907-6927      + | Olu_OL01G00020
    #>      [3] Olu_Chr_1     7085-9160      + | Olu_OL01G00030
    #>      [4] Olu_Chr_1    9830-11480      + | Olu_OL01G00040
    #>      [5] Olu_Chr_1   11467-12599      - | Olu_OL01G00050
    #>      ...       ...           ...    ... .            ...
    #>   [1899] Olu_Chr_3 977435-977752      - | Olu_OL03G05910
    #>   [1900] Olu_Chr_3 978702-978911      - | Olu_OL03G05920
    #>   [1901] Olu_Chr_3 979281-981320      - | Olu_OL03G05930
    #>   [1902] Olu_Chr_3 981778-982314      + | Olu_OL03G05940
    #>   [1903] Olu_Chr_3 982498-982746      + | Olu_OL03G05950
    #>   -------
    #>   seqinfo: 3 sequences from an unspecified genome; no seqlengths
    #>
    #> $OspRCC809
    #> GRanges object with 1433 ranges and 1 metadata column:
    #>           seqnames        ranges strand |                 gene
    #>              <Rle>     <IRanges>  <Rle> |          <character>
    #>      [1] Osp_chr_1      321-1142      - | Osp_ORCC809_01G00010
    #>      [2] Osp_chr_1     1463-2089      + | Osp_ORCC809_01G00020
    #>      [3] Osp_chr_1     2162-3370      - | Osp_ORCC809_01G00030
    #>      [4] Osp_chr_1     3774-4424      - | Osp_ORCC809_01G00040
    #>      [5] Osp_chr_1     4693-9924      - | Osp_ORCC809_01G00050
    #>      ...       ...           ...    ... .                  ...
    #>   [1429] Osp_chr_3 504915-506198      - | Osp_ORCC809_03G02980
    #>   [1430] Osp_chr_3 506377-507510      + | Osp_ORCC809_03G02990
    #>   [1431] Osp_chr_3 507856-511323      + | Osp_ORCC809_03G03000
    #>   [1432] Osp_chr_3 511533-513155      - | Osp_ORCC809_03G03010
    #>   [1433] Osp_chr_3 513841-514707      + | Osp_ORCC809_03G03020
    #>   -------
    #>   seqinfo: 3 sequences from an unspecified genome; no seqlengths

# 6 Synteny network inference

Now that we have our processed data, we can infer the synteny network. To detect synteny, we need the tabular output from BLASTp (Altschul et al. 1997) or similar programs. To get that, you can use the functions `run_diamond()`, or `run_last()`, which runs DIAMOND (Buchfink, Reuter, and Drost 2021) or last (Kiełbasa et al. 2011) from the R session and automatically parses its output to a list of data frames.1 1 1 Alternative: if you want to use a different program for similarity searches, you can run it on the command line, save the output in a DIAMOND/BLAST-like tabular format, and read the output files as a list of data frames with the function `read_diamond()` or `read_last()` (see the FAQ for details).

Let’s demonstrate how `run_diamond()` works. Needless to say, you need to have DIAMOND installed in your machine and in your PATH to run this function. To check if you have DIAMOND installed, use the function `diamond_is_installed()`.2 2 2 Note: in the code chunk below, the if statement is not required. We just added it to make sure that the function `run_diamond()` is only executed if DIAMOND is installed, to avoid problems when building this vignette in machines that do not have DIAMOND installed. If you want to reproduce the code in this vignette and do not have DIAMOND installed, you can use the example output of `run_diamond()` stored in the blast_list object (loaded with `data(blast_list)`).

    data(blast_list)
    if(diamond_is_installed()) {
        blast_list <- run_diamond(seq = pdata$seq)
    }

The output of `run_diamond()` is a list of data frames containing the tabular output of all-vs-all DIAMOND searches. Let’s take a look at it.

    # List names
    names(blast_list)
    #> [1] "Olucimarinus_Olucimarinus" "Olucimarinus_OspRCC809"
    #> [3] "OspRCC809_Olucimarinus"    "OspRCC809_OspRCC809"

    # Inspect first data frame
    head(blast_list$Olucimarinus_Olucimarinus)
    #>            query             db perc_identity length mismatches gap_open qstart
    #> 1 Olu_OL01G00010 Olu_OL01G00010           100    910          0        0      1
    #> 3 Olu_OL01G00020 Olu_OL01G00020           100    788          0        0      1
    #> 5 Olu_OL01G00030 Olu_OL01G00030           100    617          0        0      1
    #> 6 Olu_OL01G00040 Olu_OL01G00040           100    546          0        0      1
    #> 7 Olu_OL01G00050 Olu_OL01G00050           100    318          0        0      1
    #> 8 Olu_OL01G00060 Olu_OL01G00060           100    361          0        0      1
    #>   qend tstart tend    evalue bitscore
    #> 1  910      1  910  0.00e+00     1623
    #> 3  788      1  788  0.00e+00     1363
    #> 5  617      1  617  0.00e+00     1046
    #> 6  546      1  546  0.00e+00     1035
    #> 7  318      1  318 2.98e-219      595
    #> 8  361      1  361 1.72e-267      721

Now, we can use this list of DIAMOND data frames to detect synteny. Here, we reimplemented the popular MCScanX algorithm (Wang et al. 2012), originally written in C++, using the Rcpp (Eddelbuettel and François 2011) framework for R and C++ integration. This means that syntenet comes with a native version of the MCScanX algorithm, so you can run MCScanX in R without having to install it yourself. Amazing, huh?

To detect synteny and infer the synteny network, use the function `infer_syntenet()`. The output is a network represented as a so-called edge list (i.e., a 2-column data frame with node 1 and node 2 in columns 1 and 2, respectively).

    # Infer synteny network
    net <- infer_syntenet(blast_list, pdata$annotation)

    # Look at the output
    head(net)
    #>          Anchor1              Anchor2
    #> 1 Olu_OL01G00100 Osp_ORCC809_01G06480
    #> 2 Olu_OL01G00130 Osp_ORCC809_01G06440
    #> 3 Olu_OL01G00150 Osp_ORCC809_01G06420
    #> 4 Olu_OL01G00160 Osp_ORCC809_01G06410
    #> 5 Olu_OL01G00170 Osp_ORCC809_01G06400
    #> 6 Olu_OL01G00180 Osp_ORCC809_01G06390

In a synteny network, each row of the edge list represents an anchor pair. In the edge list above, for example, the genes Olu_OL01G00100 and Osp_ORCC809_01G06480 are an anchor pair (i.e., duplicates derived from a large-scale duplication event).

Note that gene IDs are preceded by IDs created with `process_input()`. Under the hood, `process_input()` uses the function `create_species_id_table()` to create unique IDs from the names of the seq and annotation lists. To obtain a data frame of all IDs and their corresponding species, you can use the following code:

    # Get a 2-column data frame of species IDs and names
    id_table <- create_species_id_table(names(proteomes))

    id_table
    #>   species_id species_name
    #> 1        Olu Olucimarinus
    #> 2        Osp    OspRCC809

# 7 Phylogenomic profiling

After inferring the synteny network, the first thing you would want to do is cluster your network and identify which phylogenetic groups are contained in each cluster. This is what we call phylogenomic profiling. This way, you can identify clade-specific clusters, and highly conserved clusters, for instance. Here, we will use an example network of BUSCO genes for 25 eudicot species, which was obtained from Zhao and Schranz (2019).

To obtain the phylogenomic profiles, you first need to cluster your network. This can be done with `cluster_network()`.3 3 3 Friendly tip: syntenet uses the Infomap algorithm to cluster networks, which has been shown to have the best performance (Zhao and Schranz 2019). However, you can use any other network clustering method implemented in the cluster_ family of functions from the igraph package by passing the function directly to the clust_function parameter (see `?cluster_network` for details). Importantly, the Infomap algorithm (default clustering method) assigns each gene to a single cluster. However, for some cases (e.g., detection of tandem arrays), you may want to use an algorithm that allows community overlap (i.e., a gene can be part of more than one cluster). If this is your case, we recommend the clique percolation algorithm, which is implemented in the R package CliquePercolation (Lange 2021).

    # Load example data and take a look at it
    data(network)
    head(network)
    #>       node1           node2
    #> 1 cca_23646  Lang_109327134
    #> 2 cca_23646  Lang_109328075
    #> 3 cca_23646   Mnot_21394516
    #> 4 cca_23646  Zjuj_107413994
    #> 5 cca_23646 adu_Aradu.8SN53
    #> 6 cca_23646     car_14082.1

    # Cluster network
    clusters <- cluster_network(network)
    head(clusters)
    #>        Gene Cluster
    #> 1 cca_23646       1
    #> 2 cca_23668       2
    #> 3 cca_32926       3
    #> 4 cca_26186       4
    #> 5 cca_24381       5
    #> 6 cca_24396       6

Now that each gene has been assigned to a cluster, we can identify the phylogenomic profiles of each cluster. This function returns a matrix of phylogenomic profiles, with clusters in rows and species in columns.

    # Phylogenomic profiling
    profiles <- phylogenomic_profile(clusters)

    # Exploring the output
    head(profiles)
    #>
    #>     Lang Mnot Zjuj adu car cca fve gma hlu jcu lja lus mdo mes mtr pbr pmu ppe
    #>   1    2    1    1   1   1   1   1   2   0   0   1   2   2   1   1   3   1   1
    #>   2    1    1    1   1   1   1   1   1   0   0   0   1   1   1   1   1   1   1
    #>   3    1    1    1   0   1   1   1   1   0   0   1   2   1   1   1   1   1   1
    #>   4    2    1    1   0   1   1   1   2   0   1   1   2   0   2   1   1   1   1
    #>   5    0    1    1   1   1   1   1   2   0   1   0   0   1   1   1   1   1   1
    #>   6    2    1    1   2   1   2   1   3   0   1   0   2   2   1   2   2   1   1
    #>
    #>     ptr pvu rco roc tpr van vra
    #>   1   1   1   1   1   1   1   1
    #>   2   1   1   0   0   1   1   1
    #>   3   1   1   1   1   1   0   0
    #>   4   0   1   1   0   1   1   0
    #>   5   1   1   1   1   1   1   2
    #>   6   2   1   1   1   1   0   1

As a plot is worth a thousand words (or numbers), you can use the function `plot_profiles()` to visualize the phylogenomic profiles as a heatmap with species in rows and synteny network clusters in columns. The heatmap generated by this function is highly customizable by users. Some important remarks are:

1. You can add a legend for species metadata (e.g., taxonomic information) by passing a 2-column data frame to the parameter species_annotation.

2. Columns (network clusters) are grouped with Ward’s clustering on a matrix of distances. The method to compute the distance matrix can be defined by users in parameters dist_function and dist_params. By default, it uses the function `stats::dist()` with parameter `method = "euclidean"`. Likewise, the function to cluster the distance matrix and additional parameters can be specified in clust_function and clust_params. By default, it uses `stats::hclust` with parameter `method = "ward.D"`.

3. The order in which species are displayed can be defined by users in parameter cluster_species. We strongly recommend passing a vector of species order that matches the species tree, so that patterns can be explored in a phylogenetic context. Importantly, if the character vector is named, vector names will be used as species names in the plot. This a nice way to replace species abbreviations with their full names.

Here, to briefly demonstrate how to play with the parameters we just mentioned in the 3 remarks above, we will:

- Create a vector with the order in which we want species to be displayed, with longer species names in vector names.

- Create a metadata data frame containing the family of each species.

- Use the function `dsvdis()` from the labdsv package to calculate Ruzicka distances when clustering columns.

    # 1) Create a named vector of custom species order to plot
    species_order <- setNames(
        # vector elements
        c(
            "vra", "van", "pvu", "gma", "cca", "tpr", "mtr", "adu", "lja",
            "Lang", "car", "pmu", "ppe", "pbr", "mdo", "roc", "fve",
            "Mnot", "Zjuj", "jcu", "mes", "rco", "lus", "ptr"
        ),
        # vector names
        c(
            "V. radiata", "V. angularis", "P. vulgaris", "G. max", "C. cajan",
            "T. pratense", "M. truncatula", "A. duranensis", "L. japonicus",
            "L. angustifolius", "C. arietinum", "P. mume", "P. persica",
            "P. bretschneideri", "M. domestica", "R. occidentalis",
            "F. vesca", "M. notabilis", "Z. jujuba",
            "J. curcas", "M. esculenta", "R. communis",
            "L. usitatissimum", "P. trichocarpa"
        )
    )
    species_order
    #>        V. radiata      V. angularis       P. vulgaris            G. max
    #>             "vra"             "van"             "pvu"             "gma"
    #>          C. cajan       T. pratense     M. truncatula     A. duranensis
    #>             "cca"             "tpr"             "mtr"             "adu"
    #>      L. japonicus  L. angustifolius      C. arietinum           P. mume
    #>             "lja"            "Lang"             "car"             "pmu"
    #>        P. persica P. bretschneideri      M. domestica   R. occidentalis
    #>             "ppe"             "pbr"             "mdo"             "roc"
    #>          F. vesca      M. notabilis         Z. jujuba         J. curcas
    #>             "fve"            "Mnot"            "Zjuj"             "jcu"
    #>      M. esculenta       R. communis  L. usitatissimum    P. trichocarpa
    #>             "mes"             "rco"             "lus"             "ptr"

    # 2) Create a metadata data frame containing the family of each species
    species_annotation <- data.frame(
        Species = species_order,
        Family = c(
            rep("Fabaceae", 11), rep("Rosaceae", 6),
            "Moraceae", "Ramnaceae", rep("Euphorbiaceae", 3),
            "Linaceae", "Salicaceae"
        )
    )
    head(species_annotation)
    #>              Species   Family
    #> V. radiata       vra Fabaceae
    #> V. angularis     van Fabaceae
    #> P. vulgaris      pvu Fabaceae
    #> G. max           gma Fabaceae
    #> C. cajan         cca Fabaceae
    #> T. pratense      tpr Fabaceae

    # 3) Plot phylogenomic profiles, but using Ruzicka distances
    plot_profiles(
        profiles,
        species_annotation,
        cluster_species = species_order,
        dist_function = labdsv::dsvdis,
        dist_params = list(index = "ruzicka")
    )

The heatmap is a nice way to observe patterns. For instance, you can see some Rosaceae-specific clusters, Fabaceae-specific clusters, and highly conserved ones as well.

If you want to explore in more details the group-specific clusters, you can use the function `find_GS_clusters()`. For that, you only need to input the profiles matrix and a data frame of species annotation (i.e., species groups).

    # Find group-specific clusters
    gs_clusters <- find_GS_clusters(profiles, species_annotation)
    #> Could not find annotation for species:
    #> hlu

    head(gs_clusters)
    #>        Group Percentage Cluster
    #> 2   Fabaceae      90.91    1156
    #> 21  Fabaceae      81.82    1170
    #> 5  Ramnaceae     100.00    1278
    #> 22  Fabaceae      90.91    1304
    #> 23  Fabaceae      81.82    1308
    #> 24  Fabaceae      90.91    1309

    # How many family-specific clusters are there?
    nrow(gs_clusters)
    #> [1] 414

As you can see, there are 414 family-specific clusters in the network. Let’s plot a heatmap of group-specific clusters only.

    # Filter profiles matrix to only include group-specific clusters
    idx <- rownames(profiles) %in% gs_clusters$Cluster
    p_gs <- profiles[idx, ]

    # Plot heatmap
    plot_profiles(
        p_gs, species_annotation,
        cluster_species = species_order,
        cluster_columns = TRUE
    )

Pretty cool, huh? You can also visualize clusters as a network plot with the function `plot_network()`. For example, let’s visualize the group-specific clusters.

    # 1) Visualize a network of first 5 GS-clusters
    id <- gs_clusters$Cluster[1:5]
    plot_network(network, clusters, cluster_id = id)

    # 2) Coloring nodes by family
    genes <- unique(c(network$node1, network$node2))
    gene_df <- data.frame(
        Gene = genes,
        Species = unlist(lapply(strsplit(genes, "_"), head, 1))
    )
    gene_df <- merge(gene_df, species_annotation)[, c("Gene", "Family")]
    head(gene_df)
    #>             Gene   Family
    #> 1 Lang_109343937 Fabaceae
    #> 2 Lang_109342839 Fabaceae
    #> 3 Lang_109356231 Fabaceae
    #> 4 Lang_109349826 Fabaceae
    #> 5 Lang_109342812 Fabaceae
    #> 6 Lang_109347788 Fabaceae

    plot_network(network, clusters, cluster_id = id, color_by = gene_df)

    # 3) Interactive visualization (zoom out and in to explore it)
    plot_network(
        network, clusters, cluster_id = id,
        interactive = TRUE, dim_interactive = c(500, 300)
    )

# 8 Microsynteny-based phylogeny reconstruction

Finally, you can use the information on presence/absence of clusters in each species to reconstruct a microsynteny-based phylogeny.

To do that, you first need to binarize the profiles matrix (0s and 1s representing absence and presence, respectively) and transpose it. This can be done with `binarize_and_tranpose()`.

    bt_mat <- binarize_and_transpose(profiles)

    # Looking at the first 5 rows and 5 columns of the matrix
    bt_mat[1:5, 1:5]
    #>
    #>        1 2 3 4 5
    #>   Lang 1 1 1 1 0
    #>   Mnot 1 1 1 1 1
    #>   Zjuj 1 1 1 1 1
    #>   adu  1 1 0 0 1
    #>   car  1 1 1 1 1

Now, you can use this transposed binary matrix as input to IQ-TREE (Minh et al. 2020) to infer a phylogeny. To do so, you can use the function `infer_microsynteny_phylogeny()`, which allows you to run IQ-TREE from an R session.4 4 4 Alternative: if you want to use a different program rather than IQ-TREE, you can use the function `profiles2phylip()` to write the transposed binary matrix to a PHYLIP file and run your favorite program on the command line. However, when inferring a phylogeny from phylogenomic profiles, you need to make sure that the program you are using supports substitution models for binary data. In IQ-TREE, for instance, using binary, morphological models requires passing parameters `-st MORPH`. You need to have IQ-TREE installed in your machine and in your PATH to run this function. You can check if you have IQ-TREE installed with `iqtree_is_installed()`.

For the sake of demonstration, we will infer a phylogeny with `infer_microsynteny_phylogeny()` using the profiles for BUSCO genes for six legume species only. We will also remove non-variable sites (i.e., clusters that are present in all species or absent in all species). However, we’re only using this filtered data set for speed issues. For real-life applications, the best thing to do is to include phylogenomic profiles for all genes (not only BUSCO genes).

    # Leave only 6 legume species and P. mume as an outgroup for testing purposes
    included <- c("gma", "pvu", "vra", "van", "cca", "pmu")
    bt_mat <- bt_mat[rownames(bt_mat) %in% included, ]

    # Remove non-variable sites
    bt_mat <- bt_mat[, colSums(bt_mat) != length(included)]

    if(iqtree_is_installed()) {
        phylo <- infer_microsynteny_phylogeny(bt_mat, outgroup = "pmu",
                                              threads = 1)
    }

The output of `infer_microsynteny_phylogeny()` is a character vector with paths to the output files from IQ-TREE. Usually, you are interested in the file ending in .treefile. This is the species tree in Newick format, and it can be visualized with your favorite tree viewer. We strongly recommend using the `read.tree()` function from the Bioconductor package treeio (Wang et al. 2020) to read the tree, and visualizing it with the ggtree Bioc package (Yu et al. 2017). For example, let’s visualize a microsynteny-based phylogeny for 123 angiosperm species, whose phylogenomic profiles were obtained from Zhao et al. (2021).

    data(angiosperm_phylogeny)

    # Plotting the tree
    library(ggtree)
    ggtree(angiosperm_phylogeny) +
        geom_tiplab(size = 3) +
        xlim(0, 0.3)

# FAQ

## How do I execute an external dependency that is not in my PATH?

If you have DIAMOND and/or IQ-TREE installed, but in a directory that is not in your PATH, you can add this given directory to your PATH with the function `Sys.setenv()`.

For example, suppose your DIAMOND binary is in `/home/username/bioinfo_tools`. To add this directory to your PATH, you would run:

    # Add example directory /home/username/bioinfo_tools to PATH
    Sys.setenv(
        PATH = paste(
            Sys.getenv("PATH"), "/home/username/bioinfo_tools", sep = ":"
        )
    )

Note that your R PATH is not the same as your system’s PATH. Thus, even if you add the directory `/home/username/bioinfo_tools` to your system’s path (e.g., by editing your ~/.bashrc file if you are on Linux), you would still need to update your R PATH.

## Can I run the DIAMOND searches on the command line and import the results?

Yes. This case is quite common for users who have a large amount of data and want to execute DIAMOND or any similarity search program in an HPC cluster (by submitting a job with `qsub` to execute a Bash script).

To do that, you will have to follow the 3 steps below:

1. Export the processed sequences (as returned by `process_input()`) with the function `export_sequences()`. This function will write the sequences to FASTA files in the directory specified in the outdir parameter.

2. Navigate (i.e., `cd`) to the directory specified in outdir, where the FASTA files are, and execute all-vs-all similarity searches. The output files must be named "[species]_[species].tsv“, where [species] indicates the basename of the FASTA files (e.g.,”speciesA" for a FASTA file named speciesA.fasta). For DIAMOND, you can use the following code (with adaptations, if you prefer):

    #!/bin/bash

    # Create output directories `dbs` and `results`
    mkdir -p dbs
    mkdir -p results

    # 1. Make dbs for each species
    for seqfile in *.fasta
    do
        dbfile="dbs/$(basename "$seqfile" .fasta)"
        diamond makedb --in "$seqfile" -d "$dbfile" --quiet
    done

    # 2. Perform all-vs-all pairwise similarity searches
    species=( $(basename -s .fasta *.fasta) )
    for (( i=0; i<${#species[@]}; i++ ))
    do
        query="${species[$i]}.fasta"
        for (( j=0; j<${#species[@]}; j++ ))
        do
            db="dbs/${species[$j]}"
            outfile="results/${species[$i]}_${species[$j]}.tsv"
            diamond blastp -q "$query" -d "$db" -o "$outfile" \
              --max-hsps 1 -k 5 --quiet
        done
    done

3. Read the output of the similarity searches into a list of data frames with the `read_diamond()` function. As input, `read_diamond()` takes the path to the directory containing the DIAMOND/BLAST output files.

## My sequence names do not match gene IDs in the annotation. What should I do?

When the names of your sequences (equivalent to FASTA headers) do not match the gene IDs in your `GRanges` objects, syntenet throws the following error:

> Sequence names in ‘seq’ do not match gene names in ‘annotation’.

In most (if not all) of the cases, this error happens because users have protein IDs as sequence names, and syntenet looks for gene IDs (i.e., using rows of the `GRanges` objects for which the column type is “gene”). This is the case, for instance, for data obtained from NCBI’s RefSeq database. To solve the issue, you need to replace protein/transcript IDs with gene IDs in sequence names, which can be done with the function `collapse_protein_ids()`.

To demonstrate how this works, let’s explore an example data set containing protein sequences and gene annotation obtained from RefSeq. The data contains information on a subset of 16 genes from the fish species Alosa alosa, and it is stored in the `extdata/RefSeq_parsing_example` directory of this package.

    # Path to directory containing data
    data_dir <- system.file(
        "extdata", "RefSeq_parsing_example", package = "syntenet"
    )
    dir(data_dir)
    #> [1] "Aalosa.fa.gz"   "Aalosa.gff3.gz"

    # Reading the files to a format that syntenet understands
    seqs <- fasta2AAStringSetlist(data_dir)
    annot <- gff2GRangesList(data_dir)

    # Taking a look at the data
    seqs
    #> $Aalosa
    #> AAStringSet object of length 21:
    #>      width seq                                              names
    #>  [1]   189 MTLKMAATMARCFPALVRPLSTR...KRRNWYLNDPYLKYQDRVNPRW XP_048084156.1 39...
    #>  [2]   348 MNEVNRTRLDTDGVRDISSPRRR...HVPYGIAQKVTHKPSISNTAAK XP_048095207.1 cy...
    #>  [3]   177 MPQDLPPAAAVPQGSMPPGKMPH...PALRRRARLRRLVTPEPLYAGW XP_048096060.1 un...
    #>  [4]   176 MPQDLPPAAAVPQGSMPPGKMPH...KERPAGRFYGLPCDAEPVYAGW XP_048096122.1 un...
    #>  [5]   137 MITKAMEVDYKLEDPKAPLEESS...GYRDAFGNELLRRLERLQQNAQ XP_048099323.1 GS...
    #>  ...   ... ...
    #> [17]   224 MGLFGKTSEKPPKDLINEWSLKI...EEEEEEEEDIEEMQTRLAALRS XP_048116262.1 ch...
    #> [18]   176 MSLVRGIMLSATRSKAWSVGRAS...QVLKNPQADHGCSCGSSFSVKL XP_048117248.1 ir...
    #> [19]   149 MAIREVCVLLLPLLALAYAEHVK...WELRDDNNKDLFCIKFPVQIVS XP_048117257.1 NP...
    #> [20]   149 MAIREVCVLLLPLLALAYAEHVK...WELRDDNNKDLFCIKFPVQIVS XP_048117266.1 NP...
    #> [21]   886 MSTSIGDKIEDFKVLTLLGKGSF...VKEKLQCLSSILGLLAGPAARR XP_048120688.1 LO...
    head(names(seqs$Aalosa))
    #> [1] "XP_048084156.1 39S ribosomal protein L35, mitochondrial [Alosa alosa]"
    #> [2] "XP_048095207.1 cytosolic 5'-nucleotidase 1A [Alosa alosa]"
    #> [3] "XP_048096060.1 uncharacterized protein si:dkey-21a6.5 isoform X1 [Alosa alosa]"
    #> [4] "XP_048096122.1 uncharacterized protein si:dkey-21a6.5 isoform X2 [Alosa alosa]"
    #> [5] "XP_048099323.1 GSK3-beta interaction protein isoform X1 [Alosa alosa]"
    #> [6] "XP_048099396.1 GSK3-beta interaction protein isoform X2 [Alosa alosa]"

    annot
    #> GRangesList object of length 1:
    #> $Aalosa
    #> GRanges object with 226 ranges and 19 metadata columns:
    #>            seqnames        ranges strand |   source     type     score
    #>               <Rle>     <IRanges>  <Rle> | <factor> <factor> <numeric>
    #>     [1] NC_063189.1    9524-52877      - |   Gnomon     gene        NA
    #>     [2] NC_063189.1   52525-52682      - |   Gnomon     CDS         NA
    #>     [3] NC_063189.1   50374-50600      - |   Gnomon     CDS         NA
    #>     [4] NC_063189.1   38459-38639      - |   Gnomon     CDS         NA
    #>     [5] NC_063189.1   31087-31235      - |   Gnomon     CDS         NA
    #>     ...         ...           ...    ... .      ...      ...       ...
    #>   [222] NC_063189.1 788729-789007      - |   Gnomon      CDS        NA
    #>   [223] NC_063189.1 779818-779990      - |   Gnomon      CDS        NA
    #>   [224] NC_063189.1 778802-778982      - |   Gnomon      CDS        NA
    #>   [225] NC_063189.1 777663-778010      - |   Gnomon      CDS        NA
    #>   [226] NC_063189.1 777553-777626      - |   Gnomon      CDS        NA
    #>             phase                 ID                                  Dbxref
    #>         <integer>        <character>                         <CharacterList>
    #>     [1]      <NA>       gene-noxred1                        GeneID:125303464
    #>     [2]         0 cds-XP_048113188.1 GeneID:125303464,Genbank:XP_048113188.1
    #>     [3]         1 cds-XP_048113188.1 GeneID:125303464,Genbank:XP_048113188.1
    #>     [4]         2 cds-XP_048113188.1 GeneID:125303464,Genbank:XP_048113188.1
    #>     [5]         1 cds-XP_048113188.1 GeneID:125303464,Genbank:XP_048113188.1
    #>     ...       ...                ...                                     ...
    #>   [222]         2 cds-XP_048105503.1 GeneID:125298741,Genbank:XP_048105503.1
    #>   [223]         2 cds-XP_048105503.1 GeneID:125298741,Genbank:XP_048105503.1
    #>   [224]         0 cds-XP_048105503.1 GeneID:125298741,Genbank:XP_048105503.1
    #>   [225]         2 cds-XP_048105503.1 GeneID:125298741,Genbank:XP_048105503.1
    #>   [226]         2 cds-XP_048105503.1 GeneID:125298741,Genbank:XP_048105503.1
    #>                   Name       gbkey         gene   gene_biotype     partial
    #>            <character> <character>  <character>    <character> <character>
    #>     [1]        noxred1        Gene      noxred1 protein_coding        true
    #>     [2] XP_048113188.1         CDS      noxred1           <NA>        true
    #>     [3] XP_048113188.1         CDS      noxred1           <NA>        true
    #>     [4] XP_048113188.1         CDS      noxred1           <NA>        true
    #>     [5] XP_048113188.1         CDS      noxred1           <NA>        true
    #>     ...            ...         ...          ...            ...         ...
    #>   [222] XP_048105503.1         CDS LOC125298741           <NA>        <NA>
    #>   [223] XP_048105503.1         CDS LOC125298741           <NA>        <NA>
    #>   [224] XP_048105503.1         CDS LOC125298741           <NA>        <NA>
    #>   [225] XP_048105503.1         CDS LOC125298741           <NA>        <NA>
    #>   [226] XP_048105503.1         CDS LOC125298741           <NA>        <NA>
    #>             start_range             Parent                   Note
    #>         <CharacterList>    <CharacterList>        <CharacterList>
    #>     [1]          .,9524
    #>     [2]                 rna-XM_048257231.1 The sequence of the ..
    #>     [3]                 rna-XM_048257231.1 The sequence of the ..
    #>     [4]                 rna-XM_048257231.1 The sequence of the ..
    #>     [5]                 rna-XM_048257231.1 The sequence of the ..
    #>     ...             ...                ...                    ...
    #>   [222]                 rna-XM_048249546.1
    #>   [223]                 rna-XM_048249546.1
    #>   [224]                 rna-XM_048249546.1
    #>   [225]                 rna-XM_048249546.1
    #>   [226]                 rna-XM_048249546.1
    #>                      exception              inference                product
    #>                    <character>            <character>            <character>
    #>     [1]                   <NA>                   <NA>                   <NA>
    #>     [2] annotated by transcr.. similar to RNA seque.. NADP-dependent oxido..
    #>     [3] annotated by transcr.. similar to RNA seque.. NADP-dependent oxido..
    #>     [4] annotated by transcr.. similar to RNA seque.. NADP-dependent oxido..
    #>     [5] annotated by transcr.. similar to RNA seque.. NADP-dependent oxido..
    #>     ...                    ...                    ...                    ...
    #>   [222]                   <NA>                   <NA> fibroblast growth fa..
    #>   [223]                   <NA>                   <NA> fibroblast growth fa..
    #>   [224]                   <NA>                   <NA> fibroblast growth fa..
    #>   [225]                   <NA>                   <NA> fibroblast growth fa..
    #>   [226]                   <NA>                   <NA> fibroblast growth fa..
    #>             protein_id transl_except
    #>            <character>   <character>
    #>     [1]           <NA>          <NA>
    #>     [2] XP_048113188.1          <NA>
    #>     [3] XP_048113188.1          <NA>
    #>     [4] XP_048113188.1          <NA>
    #>     [5] XP_048113188.1          <NA>
    #>     ...            ...           ...
    #>   [222] XP_048105503.1          <NA>
    #>   [223] XP_048105503.1          <NA>
    #>   [224] XP_048105503.1          <NA>
    #>   [225] XP_048105503.1          <NA>
    #>   [226] XP_048105503.1          <NA>
    #>   -------
    #>   seqinfo: 1 sequence from an unspecified genome; no seqlengths

The first problem we can observe is that sequence names have additional text describing the sequences (e.g., “GSK3-beta…”), and we must have only the IDs. To solve this issue, we can remove whitespace and everything that comes after it.

    # Remove whitespace and everything after it
    names(seqs$Aalosa) <- gsub(" .*", "", names(seqs$Aalosa))

    # Taking a look at the new names
    head(names(seqs$Aalosa))
    #> [1] "XP_048084156.1" "XP_048095207.1" "XP_048096060.1" "XP_048096122.1"
    #> [5] "XP_048099323.1" "XP_048099396.1"

Great, we removed the unnecessary text! However, there is still a problem: sequence names start with XP_….. If you look closer at the first row of `annot$Aalosa` (which contains ranges for genes), you will notice that none of the columns contain such XP_… IDs. This is because RefSeq uses such IDs for CDS, not for genes. Let’s check if we can indeed find the XP_… IDs in rows that have “CDS” in the `type` column.

    # Show only rows for which `type` is "CDS"
    head(annot$Aalosa[annot$Aalosa$type == "CDS"])
    #> GRanges object with 6 ranges and 19 metadata columns:
    #>          seqnames        ranges strand |   source     type     score     phase
    #>             <Rle>     <IRanges>  <Rle> | <factor> <factor> <numeric> <integer>
    #>   [1] NC_063189.1   52525-52682      - |   Gnomon      CDS        NA         0
    #>   [2] NC_063189.1   50374-50600      - |   Gnomon      CDS        NA         1
    #>   [3] NC_063189.1   38459-38639      - |   Gnomon      CDS        NA         2
    #>   [4] NC_063189.1   31087-31235      - |   Gnomon      CDS        NA         1
    #>   [5] NC_063189.1     9524-9713      - |   Gnomon      CDS        NA         2
    #>   [6] NC_063189.1 229429-229590      - |   Gnomon      CDS        NA         0
    #>                       ID                                  Dbxref           Name
    #>              <character>                         <CharacterList>    <character>
    #>   [1] cds-XP_048113188.1 GeneID:125303464,Genbank:XP_048113188.1 XP_048113188.1
    #>   [2] cds-XP_048113188.1 GeneID:125303464,Genbank:XP_048113188.1 XP_048113188.1
    #>   [3] cds-XP_048113188.1 GeneID:125303464,Genbank:XP_048113188.1 XP_048113188.1
    #>   [4] cds-XP_048113188.1 GeneID:125303464,Genbank:XP_048113188.1 XP_048113188.1
    #>   [5] cds-XP_048113188.1 GeneID:125303464,Genbank:XP_048113188.1 XP_048113188.1
    #>   [6] cds-XP_048105299.1 GeneID:125298558,Genbank:XP_048105299.1 XP_048105299.1
    #>             gbkey        gene gene_biotype     partial     start_range
    #>       <character> <character>  <character> <character> <CharacterList>
    #>   [1]         CDS     noxred1         <NA>        true
    #>   [2]         CDS     noxred1         <NA>        true
    #>   [3]         CDS     noxred1         <NA>        true
    #>   [4]         CDS     noxred1         <NA>        true
    #>   [5]         CDS     noxred1         <NA>        true          .,9524
    #>   [6]         CDS       atg2b         <NA>        <NA>
    #>                   Parent                   Note              exception
    #>          <CharacterList>        <CharacterList>            <character>
    #>   [1] rna-XM_048257231.1 The sequence of the .. annotated by transcr..
    #>   [2] rna-XM_048257231.1 The sequence of the .. annotated by transcr..
    #>   [3] rna-XM_048257231.1 The sequence of the .. annotated by transcr..
    #>   [4] rna-XM_048257231.1 The sequence of the .. annotated by transcr..
    #>   [5] rna-XM_048257231.1 The sequence of the .. annotated by transcr..
    #>   [6] rna-XM_048249342.1 The sequence of the .. unclassified transla..
    #>                    inference                product     protein_id
    #>                  <character>            <character>    <character>
    #>   [1] similar to RNA seque.. NADP-dependent oxido.. XP_048113188.1
    #>   [2] similar to RNA seque.. NADP-dependent oxido.. XP_048113188.1
    #>   [3] similar to RNA seque.. NADP-dependent oxido.. XP_048113188.1
    #>   [4] similar to RNA seque.. NADP-dependent oxido.. XP_048113188.1
    #>   [5] similar to RNA seque.. NADP-dependent oxido.. XP_048113188.1
    #>   [6]                   <NA> LOW QUALITY PROTEIN:.. XP_048105299.1
    #>       transl_except
    #>         <character>
    #>   [1]          <NA>
    #>   [2]          <NA>
    #>   [3]          <NA>
    #>   [4]          <NA>
    #>   [5]          <NA>
    #>   [6]          <NA>
    #>   -------
    #>   seqinfo: 1 sequence from an unspecified genome; no seqlengths

The XP_… IDs can be found in the `Name` column. Note also that the gene IDs, which are what we need for syntenet, are in the `gene` column. To collapse protein IDs to gene IDs with `collapse_protein_ids()`, we will need to create a list of 2-column data frames containing the correspondence between protein IDs and gene IDs for each species. In this example, we can do that by extracting the columns `Name` and `gene` from rows that represent CDS ranges.

    # Create a list of data frames containing protein-to-gene ID correspondences
    protein2gene <- lapply(annot, function(x) {

        # Extract only CDS ranges
        cds_ranges <- x[x$type == "CDS"]

        # Create the ID correspondence data frame
        df <- data.frame(
            protein_id = cds_ranges$Name,
            gene_id = cds_ranges$gene
        )

        # Remove duplicate rows
        df <- df[!duplicated(df$protein_id), ]

        return(df)
    })

    # Taking a look at the list
    protein2gene
    #> $Aalosa
    #>         protein_id        gene_id
    #> 1   XP_048113188.1        noxred1
    #> 6   XP_048105299.1          atg2b
    #> 47  XP_048099323.1   LOC125294505
    #> 50  XP_048099476.1   LOC125294505
    #> 52  XP_048099396.1   LOC125294505
    #> 54  XP_048099557.1   LOC125294505
    #> 56  XP_048117257.1   LOC125306123
    #> 60  XP_048117266.1   LOC125306123
    #> 64  XP_048117248.1          isca2
    #> 68  XP_048105318.1           eml5
    #> 119 XP_048103211.1         itpk1b
    #> 130 XP_048115651.1   LOC125305081
    #> 132 XP_048120688.1           plk4
    #> 149 XP_048105359.1         hspa4l
    #> 168 XP_048116262.1          chmp3
    #> 176 XP_048111661.1          reep1
    #> 183 XP_048084156.1         mrpl35
    #> 187 XP_048096060.1 si:dkey-21a6.5
    #> 192 XP_048096122.1 si:dkey-21a6.5
    #> 197 XP_048095207.1        nt5c1ab
    #> 203 XP_048105503.1   LOC125298741

Finally, we can pass the list of sequences and the list of ID correspondences to `collapse_protein_ids()`, which will return a list of `AAStringSet` objects with gene IDs in sequence names.

    # Collapse protein IDs to gene IDs in list of sequences
    new_seq <- collapse_protein_ids(seqs, protein2gene)

    # Looking at the new sequences
    new_seq
    #> $Aalosa
    #> AAStringSet object of length 16:
    #>      width seq                                              names
    #>  [1]  1946 MADRTAPNCHLRLEWVYGYRGHQ...YVISAGGDDRSLFVWKCVHTPH eml5
    #>  [2]  1882 MPWPFSESIKKRACRYLLHRYLG...MRNQIQPDARQEETQKWRLGEE atg2b
    #>  [3]   886 MSTSIGDKIEDFKVLTLLGKGSF...VKEKLQCLSSILGLLAGPAARR plk4
    #>  [4]   769 MSVVGFDVGFQNCYIAVARSGGI...GGDAKSGGSSHQPAAPGEMEVE hspa4l
    #>  [5]   480 MAAPAYVFGQKVWLSARISPLRV...HTHTHAHLESKVHQHQHIHFQC LOC125298741
    #>  ...   ... ...
    #> [12]   189 MTLKMAATMARCFPALVRPLSTR...KRRNWYLNDPYLKYQDRVNPRW mrpl35
    #> [13]   177 MPQDLPPAAAVPQGSMPPGKMPH...PALRRRARLRRLVTPEPLYAGW si:dkey-21a6.5
    #> [14]   176 MSLVRGIMLSATRSKAWSVGRAS...QVLKNPQADHGCSCGSSFSVKL isca2
    #> [15]   149 MAIREVCVLLLPLLALAYAEHVK...WELRDDNNKDLFCIKFPVQIVS LOC125306123
    #> [16]   137 MITKAMEVDYKLEDPKAPLEESS...GYRDAFGNELLRRLERLQQNAQ LOC125294505

As you can see, protein IDs have been replaced with gene IDs. If there are multiple proteins for the same gene (i.e., different isoforms), the function keeps only the longest sequence (also known as protein products of the primary transcript). This way, the number of sequences will never be greater than the number of genes, which is what syntenet expects.

# Session information

This document was created under the following conditions:

    sessionInfo()
    #> R version 4.5.1 (2025-06-13 ucrt)
    #> Platform: x86_64-w64-mingw32/x64
    #> Running under: Windows Server 2022 x64 (build 20348)
    #>
    #> Matrix products: default
    #>   LAPACK version 3.12.1
    #>
    #> locale:
    #> [1] LC_COLLATE=C
    #> [2] LC_CTYPE=English_United States.utf8
    #> [3] LC_MONETARY=English_United States.utf8
    #> [4] LC_NUMERIC=C
    #> [5] LC_TIME=English_United States.utf8
    #>
    #> time zone: America/New_York
    #> tzcode source: internal
    #>
    #> attached base packages:
    #> [1] stats     graphics  grDevices utils     datasets  methods   base
    #>
    #> other attached packages:
    #> [1] ggtree_3.16.3    syntenet_1.10.2  BiocStyle_2.36.0
    #>
    #> loaded via a namespace (and not attached):
    #>   [1] bitops_1.0-9                rlang_1.1.6
    #>   [3] magrittr_2.0.3              matrixStats_1.5.0
    #>   [5] compiler_4.5.1              mgcv_1.9-3
    #>   [7] vctrs_0.6.5                 pkgconfig_2.0.3
    #>   [9] crayon_1.5.3                fastmap_1.2.0
    #>  [11] XVector_0.48.0              labeling_0.4.3
    #>  [13] Rsamtools_2.24.0            rmarkdown_2.29
    #>  [15] UCSC.utils_1.4.0            network_1.19.0
    #>  [17] purrr_1.1.0                 xfun_0.52
    #>  [19] cachem_1.1.0                aplot_0.2.8
    #>  [21] GenomeInfoDb_1.44.1         jsonlite_2.0.0
    #>  [23] DelayedArray_0.34.1         BiocParallel_1.42.1
    #>  [25] parallel_4.5.1              data.tree_1.1.0
    #>  [27] cluster_2.1.8.1             R6_2.6.1
    #>  [29] bslib_0.9.0                 stringi_1.8.7
    #>  [31] RColorBrewer_1.1-3          rtracklayer_1.68.0
    #>  [33] GenomicRanges_1.60.0        jquerylib_0.1.4
    #>  [35] Rcpp_1.1.0                  bookdown_0.43
    #>  [37] SummarizedExperiment_1.38.1 knitr_1.50
    #>  [39] IRanges_2.42.0              Matrix_1.7-3
    #>  [41] splines_4.5.1               igraph_2.1.4
    #>  [43] tidyselect_1.2.1            dichromat_2.0-0.1
    #>  [45] abind_1.4-8                 yaml_2.3.10
    #>  [47] codetools_0.2-20            curl_6.4.0
    #>  [49] lattice_0.22-7              tibble_3.3.0
    #>  [51] treeio_1.32.0               Biobase_2.68.0
    #>  [53] withr_3.0.2                 coda_0.19-4.1
    #>  [55] evaluate_1.0.4              Rtsne_0.17
    #>  [57] gridGraphics_0.5-1          Biostrings_2.76.0
    #>  [59] pillar_1.11.0               BiocManager_1.30.26
    #>  [61] MatrixGenerics_1.20.0       stats4_4.5.1
    #>  [63] ggfun_0.2.0                 generics_0.1.4
    #>  [65] RCurl_1.98-1.17             S4Vectors_0.46.0
    #>  [67] ggplot2_3.5.2               scales_1.4.0
    #>  [69] tidytree_0.4.6              ggnetwork_0.5.13
    #>  [71] glue_1.8.0                  pheatmap_1.0.13
    #>  [73] lazyeval_0.2.2              tools_4.5.1
    #>  [75] BiocIO_1.18.0               GenomicAlignments_1.44.0
    #>  [77] fs_1.6.6                    XML_3.99-0.18
    #>  [79] grid_4.5.1                  tidyr_1.3.1
    #>  [81] ape_5.8-1                   patchwork_1.3.1
    #>  [83] nlme_3.1-168                networkD3_0.4.1
    #>  [85] GenomeInfoDbData_1.2.14     restfulr_0.0.16
    #>  [87] cli_3.6.5                   intergraph_2.0-4
    #>  [89] labdsv_2.1-0                S4Arrays_1.8.1
    #>  [91] dplyr_1.1.4                 gtable_0.3.6
    #>  [93] yulab.utils_0.2.0           sass_0.4.10
    #>  [95] digest_0.6.37               BiocGenerics_0.54.0
    #>  [97] SparseArray_1.8.1           ggplotify_0.1.2
    #>  [99] rjson_0.2.23                htmlwidgets_1.6.4
    #> [101] farver_2.1.2                htmltools_0.5.8.1
    #> [103] lifecycle_1.0.4             httr_1.4.7
    #> [105] statnet.common_4.12.0       MASS_7.3-65

# References

Altschul, Stephen F, Thomas L Madden, Alejandro A Schäffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J Lipman. 1997. “Gapped Blast and Psi-Blast: A New Generation of Protein Database Search Programs.” Nucleic Acids Research 25 (17): 3389–3402.

Buchfink, Benjamin, Klaus Reuter, and Hajk-Georg Drost. 2021. “Sensitive Protein Alignments at Tree-of-Life Scale Using Diamond.” Nature Methods 18 (4): 366–68.

Eddelbuettel, Dirk, and Romain François. 2011. “Rcpp: Seamless R and C++ Integration.” Journal of Statistical Software 40: 1–18.

Kiełbasa, Szymon M, Raymond Wan, Kengo Sato, Paul Horton, and Martin C Frith. 2011. “Adaptive Seeds Tame Genomic Sequence Comparison.” Genome Research 21 (3): 487–93.

Lange, Jens. 2021. “CliquePercolation: An R Package for Conducting and Visualizing Results of the Clique Percolation Network Community Detection Algorithm.” Journal of Open Source Software 6 (62): 3210.

Minh, Bui Quang, Heiko A Schmidt, Olga Chernomor, Dominik Schrempf, Michael D Woodhams, Arndt Von Haeseler, and Robert Lanfear. 2020. “IQ-Tree 2: New Models and Efficient Methods for Phylogenetic Inference in the Genomic Era.” Molecular Biology and Evolution 37 (5): 1530–4.

Vandepoele, Klaas, Michiel Van Bel, Guilhem Richard, Sofie Van Landeghem, Bram Verhelst, Hervé Moreau, Yves Van de Peer, Nigel Grimsley, and Gwenael Piganeau. 2013. “Pico-Plaza, a Genome Database of Microbial Photosynthetic Eukaryotes.” Environmental Microbiology 15 (8): 2147–53.

Wang, Li-Gen, Tommy Tsan-Yuk Lam, Shuangbin Xu, Zehan Dai, Lang Zhou, Tingze Feng, Pingfan Guo, et al. 2020. “Treeio: An R Package for Phylogenetic Tree Input and Output with Richly Annotated and Associated Data.” Molecular Biology and Evolution 37 (2): 599–603.

Wang, Yupeng, Haibao Tang, Jeremy D DeBarry, Xu Tan, Jingping Li, Xiyin Wang, Tae-ho Lee, et al. 2012. “MCScanX: A Toolkit for Detection and Evolutionary Analysis of Gene Synteny and Collinearity.” Nucleic Acids Research 40 (7): e49–e49.

Yu, Guangchuang, David K Smith, Huachen Zhu, Yi Guan, and Tommy Tsan-Yuk Lam. 2017. “Ggtree: An R Package for Visualization and Annotation of Phylogenetic Trees with Their Covariates and Other Associated Data.” Methods in Ecology and Evolution 8 (1): 28–36.

Zhao, Tao, Rens Holmer, Suzanne de Bruijn, Gerco C Angenent, Harrold A van den Burg, and M Eric Schranz. 2017. “Phylogenomic Synteny Network Analysis of Mads-Box Transcription Factor Genes Reveals Lineage-Specific Transpositions, Ancient Tandem Duplications, and Deep Positional Conservation.” The Plant Cell 29 (6): 1278–92.

Zhao, Tao, and M Eric Schranz. 2017. “Network Approaches for Plant Phylogenomic Synteny Analysis.” Current Opinion in Plant Biology 36: 129–34.

———. 2019. “Network-Based Microsynteny Analysis Identifies Major Differences and Genomic Outliers in Mammalian and Angiosperm Genomes.” Proceedings of the National Academy of Sciences 116 (6): 2165–74.

Zhao, Tao, Arthur Zwaenepoel, Jia-Yu Xue, Shu-Min Kao, Zhen Li, M Eric Schranz, and Yves Van de Peer. 2021. “Whole-Genome Microsynteny-Based Phylogeny of Angiosperms.” Nature Communications 12 (1): 1–14.