Unspecified WGDs

[TommySalvetti01]

Hi, Dr. Lovell. I ran analysis and I got this error:

############## Flagging over-dispered OGs ...amphioxus: 2048 genes in 99 OGs hit > 8 unique places ...chicken : 246 genes in 8 OGs hit > 8 unique places ...frog : 735 genes in 24 OGs hit > 8 unique places ...human : 367 genes in 13 OGs hit > 8 unique places ...lamprey : 471 genes in 13 OGs hit > 8 unique places ...zebrafish: 1705 genes in 49 OGs hit > 8 unique places NOTE! Genomes flagged *** have > 5% of genes in over-dispersed orthogroups. These are likely not great annotations, or the synteny run contains un-specified WGDs. Regardless, these should be examined carefully

When I visualized the riparian plot, almost no syntenic connections were drawn between zebrafish and the other organisms. Could this error be the cause of this? If so, how could I solve it? And since zebrafish underwent a WGD, is there a way to specify that in GENESPACE?

[jtlovell]

What do the dotplots look like? I can't imagine there is much synteny between human and lamprey.

[jtlovell]

Also, aren't there some WGDs between mammals and fishes? You need to parameterize your run so that ploidy matches the biology of the system.

[TommySalvetti01]

Thank you for the response! I'll drop screenshots of the dotplots on this reply. There is a WGD between mammals fishes, yes. How would I go about parameterizing the run?

Here's the dotplot: when I generated it, I got the following warning: Warning message: In ggdotplot(hits = hits, type = "all", verbose = FALSE) : no syntenic hits found for human vs. lamprey

It looks like you're right.

[jtlovell]

Hmm. I think this is probably a case that GENESPACE will not be particularly useful. If you are just trying to generally show which chromosomes share some common origin, perhaps its ok, but generally we discourage folks using it when most of the genome is not collinear.

That said, you probably have some options here. First off, you should parameterize with the ploidy of the genomes relative to their MRCA. So, if there are two sequential WGDs in fish and none in mammals since they diverged, then lamprey should have a ploidy of 4 and human of 1.

Also, it looks from your plot, like there are regions of general synteny, but these are really choppy. GENESPACE can deal with this. We had a similar case in sphagnum, which has really broken up synteny in its 300Mya WGD (see fig 3 here). These are some parameters that may help:

gp <- init_genespace(
  ...,
  blkSize = 3, # decrease minimum block size
  nGaps = 10, # increase gaps within blocks
  useHOGs = FALSE, # use depregated orthogroups, which don't split as much
  synBuff = 250, # increase the region around syntenic anchors
  ...)

You might also just try two genomes and run with

gp <- init_genespace(
  ...,
  diamondUltraSens = TRUE, # use ultrasensitive search for better estimates of deep divergence
  ...)

[jtlovell]

I'm gonna close this, but feel free to reopen if anything comes up.

[TommySalvetti01]

Hi again! Thank you for all the help! I reran GENESPACE according to your suggestions. However, I ran into another error:

############################

5. Building synteny-constrained orthogroups ... ############## Running Orthofinder within syntenic regions ...zebrafish v. zebrafish: Error in rbindlist(mclapply(1:nrow(ofDirs), mc.cores = nCores, function(k) { : Item 1 of input is not a data.frame, data.table or list In addition: Warning message: In mclapply(1:nrow(ofDirs), mc.cores = nCores, function(k) { : all scheduled cores encountered errors in user code

How can I go about troubleshooting this?

[jtlovell]

Can you re-run, but setting init_genespace(..., nCores = 1, ...), then print the full output here. Also, can you share the urls for the genomes in the run so I can try to re-create it and make the error more informative.

[jtlovell]

one possibility is that you never have a syntenic region of > 40 genes (bare minimum for orthofinder to be useful). This would happen in a fully diploidized genome with super ancient WGDs.

[TommySalvetti01]

Thank you for all the responses! I downloaded the gff and CDS directly from the NCBI website. Here are the URLs:

Human: https://www.ncbi.nlm.nih.gov/assembly/GCF_000001405.40 Frog: https://www.ncbi.nlm.nih.gov/assembly/GCF_000004195.4 Chicken: https://www.ncbi.nlm.nih.gov/assembly/GCF_016699485.2 Zebrafish: https://www.ncbi.nlm.nih.gov/assembly/GCF_000002035.6 Lamprey: https://www.ncbi.nlm.nih.gov/assembly/GCF_010993605.1 Amphioxus: https://www.ncbi.nlm.nih.gov/assembly/GCA_927797965.1

For Amphioxus, the source database is GenBank. For the rest, the source database is RefSeq.

[TommySalvetti01]

And here is the output I got. Apparently there's a different error now.

gp <- init_genespace(

• wd = wd,
• ploidy = c(1, 1, 8, 4, 4, 4),
• path2mcscanx = path2mcscanx,
• blkSize = 3,
• nGaps = 10,
• useHOGs = FALSE,
• synBuff = 250,
• nCores = 1
• ) Checking Working Directory ... PASS: ~/desktop/workingDirectory Checking user-defined parameters ... Genome IDs & ploidy ... amphioxus: 1 chicken : 1 frog : 8 human : 4 lamprey : 4 zebrafish: 4 Outgroup ... NONE n. parallel processes ... 1 collinear block size ... 3 collinear block search radius ... 15 n gaps in collinear block ... 10 synteny buffer size... 250 only orthogroups hits as anchors ... TRUE n secondary hits ... 0 only orthogroup hits for homeolog block anchors ... FALSE Checking annotation files (.bed and peptide .fa): amphioxus: 17523 / 17523 geneIDs exactly match (PASS) chicken : 18093 / 18093 geneIDs exactly match (PASS) frog : 21898 / 21898 geneIDs exactly match (PASS) human : 20678 / 20678 geneIDs exactly match (PASS) lamprey : 17580 / 17580 geneIDs exactly match (PASS) zebrafish: 26508 / 26508 geneIDs exactly match (PASS) Checking dependencies ... WARNING!! DIAMOND version 0.914 is installed but OrthoFinder needs DIAMOND2. Setting path2diamond and path2orthofinder as NA. Install an up-to-date version or run OrthoFinder in an environment with DIAMOND2. WARNING!!, orthofinderInBlk was set to TRUE; however, this routine requires a valid path to the OrthoFinder program to be specified within R. Therefore, orthofinderInBlk has been set to FALSE. If this is not desired, please re-run build_params with a valid path2orthofinder. Found valid MCScanX_h executable: /Users/tommysalvetti/desktop/MCScanX/MCScanX_h out <- run_genespace(gp, overwrite = T)

############################

1. Running orthofinder (or parsing existing results) Checking for existing orthofinder results ... ... found existing run, not re-running orthofinder

############################

2. Combining and annotating bed files w/ OGs and tandem array info ... ############## Flagging chrs. w/ < 6 unique orthogroups ...amphioxus: 480 genes on 273 small chrs. ...chicken : 378 genes on 50 small chrs. ...frog : 66 genes on 47 small chrs. ...human : 7 genes on 5 small chrs. ...lamprey : 935 genes on 542 small chrs. ...zebrafish: 581 genes on 401 small chrs. NOTE! Genomes flagged have > 5% of genes on small chrs. These are likely not great assemblies and should be examined carefully ############## Flagging over-dispered OGs ...amphioxus: 0 genes in 0 OGs hit > 64 unique places ...chicken : 0 genes in 0 OGs hit > 64 unique places ...frog : 0 genes in 0 OGs hit > 64 unique places ...human : 0 genes in 0 OGs hit > 64 unique places ...lamprey : 0 genes in 0 OGs hit > 64 unique places ...zebrafish: 0 genes in 0 OGs hit > 64 unique places ############## Annotation summaries (after exclusions): ...amphioxus: 17043 genes in 12052 OGs || 3734 genes in 1306 arrays ...chicken : 17715 genes in 12426 OGs || 2981 genes in 678 arrays ...frog : 21832 genes in 13243 OGs || 5918 genes in 1234 arrays ...human : 20671 genes in 13682 OGs || 4355 genes in 1090 arrays ...lamprey : 16645 genes in 11975 OGs || 2322 genes in 685 arrays ...zebrafish: 25927 genes in 14339 OGs || 5929 genes in 1375 arrays

############################

3. Combining and annotating the blast files with orthogroup info ... ...zebrafish v. zebrafish: total hits = 349319, same og = 130367 ...zebrafish v. frog: total hits = 353416, same og = 41348 ...frog v. frog: total hits = 265194, same og = 102717 ...zebrafish v. human: total hits = 306267, same og = 36286 ...zebrafish v. lamprey: total hits = 275468, same og = 28641 ...zebrafish v. chicken: total hits = 275578, same og = 31650 ...frog v. human: total hits = 275316, same og = 35740 ...human v. human: total hits = 221074, same og = 72775 ...frog v. lamprey: total hits = 237257, same og = 25461 ...frog v. chicken: total hits = 242182, same og = 29248 ...lamprey v. lamprey: total hits = 172180, same og = 50059 ...chicken v. chicken: total hits = 190677, same og = 66994 ...human v. chicken: total hits = 224957, same og = 30085 ...human v. lamprey: total hits = 205373, same og = 22348 ...lamprey v. chicken: total hits = 185446, same og = 20519 ...zebrafish v. amphioxus: total hits = 176363, same og = 14697 ...amphioxus v. amphioxus: total hits = 122503, same og = 58772 ...frog v. amphioxus: total hits = 148749, same og = 13091 ...human v. amphioxus: total hits = 124806, same og = 12235 ...lamprey v. amphioxus: total hits = 114054, same og = 10977 ...chicken v. amphioxus: total hits = 116159, same og = 11234 ############## Generating dotplots for all hits ... Done!

############################

4. Flagging synteny for each pair of genomes ... ...zebrafish v. zebrafish: 88922 hits (27564 anchors) in 676 blocks (177 SVs, 107 regions) ...frog v. frog: 95125 hits (23304 anchors) in 658 blocks (538 SVs, 149 regions) ...human v. human: 65695 hits (21611 anchors) in 393 blocks (258 SVs, 128 regions) ...lamprey v. lamprey: 37384 hits (18829 anchors) in 1135 blocks (361 SVs, 165 regions) ...zebrafish v. frog: 6319 hits (4296 anchors) in 790 blocks (668 SVs, 477 regions) ...zebrafish v. human: 6081 hits (4188 anchors) in 767 blocks (581 SVs, 482 regions) ...frog v. human: 14299 hits (10923 anchors) in 1023 blocks (926 SVs, 520 regions) ...zebrafish v. chicken: 5855 hits (4122 anchors) in 714 blocks (581 SVs, 444 regions) ...human v. chicken: 13644 hits (11377 anchors) in 662 blocks (583 SVs, 426 regions) ...frog v. chicken: 13128 hits (10474 anchors) in 827 blocks (781 SVs, 383 regions) ...zebrafish v. lamprey: 1016 hits (503 anchors) in 152 blocks (84 SVs, 75 regions) ...frog v. lamprey: 3897 hits (1972 anchors) in 604 blocks (459 SVs, 244 regions) ...human v. lamprey: 3900 hits (1890 anchors) in 597 blocks (419 SVs, 242 regions) ...lamprey v. chicken: 2824 hits (1298 anchors) in 442 blocks (332 SVs, 142 regions) ...zebrafish v. amphioxus: 416 hits (72 anchors) in 19 blocks (8 SVs, 11 regions) ...frog v. amphioxus: 891 hits (369 anchors) in 141 blocks (102 SVs, 43 regions) ...human v. amphioxus: 922 hits (357 anchors) in 127 blocks (89 SVs, 39 regions) ...chicken v. amphioxus: 711 hits (314 anchors) in 139 blocks (104 SVs, 35 regions) ...lamprey v. amphioxus: 954 hits (503 anchors) in 203 blocks (146 SVs, 60 regions) ...chicken v. chicken: 59703 hits (18084 anchors) in 96 blocks (0 SVs, 0 regions) ...amphioxus v. amphioxus: 30028 hits (17491 anchors) in 297 blocks (0 SVs, 0 regions)

############################

5. Building synteny-constrained orthogroups ... ############## Running Orthofinder within syntenic regions ...zebrafish v. zebrafish: Error in if (file.exists(sidf)) { : argument is of length zero

[jtlovell]

Oh, well, thats a more informative issue. Did you open R from an environment without orthofinder in the path? Since you are not giving GENESPACE a path to orthofinder, it assumes its called via orthofinder. However, since this isn't valid, it won't run orthofinder and thus, errors out when you try to do orthofinderInBlk. Try re-running with a valid path to orthofinder.

[TommySalvetti01]

I reran this time by opening R in a conda environment containing orthofinder, but I got the same error again. Should I try something else?

[jtlovell]

OK - I'm gonna run it from scratch here and see what happens. More soon. FYI, using this script (taken from the tutorial)

genomeRepo <- "~/Downloads/testVerts"
gsWd <- genomeRepo
urls <- c(
  human ="GCF/000/001/405/GCF_000001405.40_GRCh38.p14/GCF_000001405.40_GRCh38.p14_",
  frog = "GCF/000/004/195/GCF_000004195.4_UCB_Xtro_10.0/GCF_000004195.4_UCB_Xtro_10.0_",
  Zebrafish = "GCF/000/002/035/GCF_000002035.6_GRCz11/GCF_000002035.6_GRCz11_",
  chicken = "GCF/016/699/485/GCF_016699485.2_bGalGal1.mat.broiler.GRCg7b/GCF_016699485.2_bGalGal1.mat.broiler.GRCg7b_",
  Lamprey = "GCF/010/993/605/GCF_010993605.1_kPetMar1.pri/GCF_010993605.1_kPetMar1.pri_",
  Amphioxus = "GCA/927/797/965/GCA_927797965.1_BraLan3/GCA_927797965.1_BraLan3_")

genomes2run <- names(urls)
urls <- file.path("https://ftp.ncbi.nlm.nih.gov/genomes/all", urls)
translatedCDS <- sprintf("%stranslated_cds.faa.gz", urls)
geneGff <- sprintf("%sgenomic.gff.gz", urls)

names(translatedCDS) <- genomes2run
names(geneGff) <- genomes2run
writeDirs <- file.path(genomeRepo, genomes2run)
names(writeDirs) <- genomes2run
for(i in genomes2run){
  print(i)
  if(!dir.exists(writeDirs[i]))
    dir.create(writeDirs[i], recursive = T)
  download.file(
    url = geneGff[i], 
    destfile = file.path(writeDirs[i], basename(geneGff[i])))
  download.file(
    url = translatedCDS[i], 
    destfile = file.path(writeDirs[i], basename(translatedCDS[i])))
}

parsedPaths <- parse_annotations(
  rawGenomeRepo = genomeRepo,
  genomeDirs = genomes2run,
  genomeIDs = genomes2run,
  presets = "ncbi",
  genespaceWd = gsWd)

gp <- init_genespace(
  wd = gsWd, 
  genomeIDs = genomes2run, 
  orthofinderInBlk = TRUE,
  nCores = 12, 
  ploidy = c(4, 8, 4, 1, 4, 1),
  path2mcscanx = "~/Desktop/programs/MCScanX/")
out <- run_genespace(gp)

[jtlovell]

what version of GENESPACE are you running? This error looks like it was solved in one of the more recent releases. This is what I get ...

############################
5. Building synteny-constrained orthogroups ...
    ##############
    Running Orthofinder within syntenic regions
    ...Zebrafish v. Zebrafish: <50% (25.7%) of syn. hits in regions with >=40 genes, not running
    ...frog v. frog:           <50% (34.3%) of syn. hits in regions with >=40 genes, not running
    ...human v. human:         <50% (19.6%) of syn. hits in regions with >=40 genes, not running
    ...Lamprey v. Lamprey:     <50% (17.8%) of syn. hits in regions with >=40 genes, not running
    ...Zebrafish v. frog:      <50% (12.5%) of syn. hits in regions with >=40 genes, not running
    ...Zebrafish v. human:     <50% (13.4%) of syn. hits in regions with >=40 genes, not running
    ...frog v. human:          n syn / syn OG / inblk OG hits = 15135 / 12271 / 12655
    ...Zebrafish v. chicken:   <50% (12.5%) of syn. hits in regions with >=40 genes, not running
    ...human v. chicken:       n syn / syn OG / inblk OG hits = 14175 / 12212 / 12467
    ...frog v. chicken:        n syn / syn OG / inblk OG hits = 13806 / 11651 / 12032
    ...Zebrafish v. Lamprey:   <50% (0%) of syn. hits in regions with >=40 genes, not running
    ...frog v. Lamprey:        <50% (17.8%) of syn. hits in regions with >=40 genes, not running
    ...human v. Lamprey:       <50% (13.6%) of syn. hits in regions with >=40 genes, not running
    ...Lamprey v. chicken:     <50% (28.1%) of syn. hits in regions with >=40 genes, not running
    ...Zebrafish v. Amphioxus: <50% (0%) of syn. hits in regions with >=40 genes, not running
    ...frog v. Amphioxus:      <50% (0%) of syn. hits in regions with >=40 genes, not running
    ...human v. Amphioxus:     <50% (0%) of syn. hits in regions with >=40 genes, not running
    ...chicken v. Amphioxus:   <50% (0%) of syn. hits in regions with >=40 genes, not running
    ...Lamprey v. Amphioxus:   <50% (0%) of syn. hits in regions with >=40 genes, not running
    ...chicken v. chicken:     no non-self blocks
    ...Amphioxus v. Amphioxus: no non-self blocks
    ##############
    Pulling syntenic orthogroups
    Done!

That said, it does error out for me later in the run. During riparian plotting. This is because there is no synteny. I'll address this when I can so it errors out more informatively.

[TommySalvetti01]

Thank you for all the help! I've been using version 1.1.4. I'll try updating GENESPACE and running it again. Fascinating that there is no synteny...

[jtlovell]

Alright. let me know if it doesn't work. closing, but feel free to re-open

[TommySalvetti01]

I attempted to your script with nCores = 1 instead of 12 (since my computer crashed both times I tried with nCores = 12) and it errored out again :( here's the complete log

out <- run_genespace(gp, overwrite = T)

############################

1. Running orthofinder (or parsing existing results) Checking for existing orthofinder results ... ... found existing run, not re-running orthofinder

############################

2. Combining and annotating bed files w/ OGs and tandem array info ... ############## Flagging chrs. w/ < 10 unique orthogroups ...Amphioxus: 519 genes on 278 small chrs. ...Lamprey : 990 genes on 549 small chrs. ...Zebrafish: 594 genes on 403 small chrs. ...chicken : 475 genes on 55 small chrs. ...frog : 66 genes on 47 small chrs. ...human : 7 genes on 5 small chrs. NOTE! Genomes flagged have > 5% of genes on small chrs. These are likely not great assemblies and should be examined carefully ############## Flagging over-dispered OGs ...Amphioxus: 144 genes in 1 OGs hit > 64 unique places ...Lamprey : 0 genes in 0 OGs hit > 64 unique places ...Zebrafish: 0 genes in 0 OGs hit > 64 unique places ...chicken : 0 genes in 0 OGs hit > 64 unique places ...frog : 0 genes in 0 OGs hit > 64 unique places ...human : 0 genes in 0 OGs hit > 64 unique places ############## Annotation summaries (after exclusions): ...Amphioxus: 16879 genes in 12123 OGs || 3269 genes in 1187 arrays ...Lamprey : 16590 genes in 11995 OGs || 2246 genes in 657 arrays ...Zebrafish: 25914 genes in 14387 OGs || 5472 genes in 1210 arrays ...chicken : 17618 genes in 12436 OGs || 2799 genes in 629 arrays ...frog : 21832 genes in 13298 OGs || 5731 genes in 1174 arrays ...human : 20671 genes in 13731 OGs || 4170 genes in 1045 arrays

############################

3. Combining and annotating the blast files with orthogroup info ... ...Zebrafish v. Zebrafish: total hits = 349329, same og = 130107 ...Zebrafish v. frog: total hits = 353417, same og = 41486 ...frog v. frog: total hits = 265197, same og = 102574 ...Zebrafish v. human: total hits = 306272, same og = 35997 ...Zebrafish v. Lamprey: total hits = 275491, same og = 28359 ...Zebrafish v. chicken: total hits = 275588, same og = 31492 ...human v. human: total hits = 221060, same og = 72279 ...frog v. human: total hits = 275348, same og = 35620 ...frog v. chicken: total hits = 242185, same og = 29018 ...frog v. Lamprey: total hits = 237256, same og = 25244 ...chicken v. chicken: total hits = 190673, same og = 66822 ...human v. chicken: total hits = 224953, same og = 29877 ...Lamprey v. Lamprey: total hits = 172180, same og = 49858 ...human v. Lamprey: total hits = 205356, same og = 22062 ...Lamprey v. chicken: total hits = 185446, same og = 20309 ...Zebrafish v. Amphioxus: total hits = 176348, same og = 14434 ...Amphioxus v. Amphioxus: total hits = 122489, same og = 58562 ...frog v. Amphioxus: total hits = 148757, same og = 12874 ...human v. Amphioxus: total hits = 124814, same og = 12023 ...chicken v. Amphioxus: total hits = 116160, same og = 11065 ...Lamprey v. Amphioxus: total hits = 114052, same og = 10760 ############## Generating dotplots for all hits ... Done!

############################

4. Flagging synteny for each pair of genomes ... ...Zebrafish v. Zebrafish: 77492 hits (27755 anchors) in 657 blocks (141 SVs, 105 regions) ...frog v. frog: 90163 hits (23286 anchors) in 444 blocks (324 SVs, 117 regions) ...human v. human: 61297 hits (21740 anchors) in 296 blocks (165 SVs, 111 regions) ...chicken v. chicken: 60833 hits (19510 anchors) in 473 blocks (292 SVs, 98 regions) ...Lamprey v. Lamprey: 36138 hits (18158 anchors) in 819 blocks (114 SVs, 73 regions) ...Zebrafish v. frog: 6611 hits (4182 anchors) in 619 blocks (499 SVs, 384 regions) ...Zebrafish v. human: 6614 hits (4184 anchors) in 610 blocks (423 SVs, 400 regions) ...frog v. human: 15139 hits (11008 anchors) in 815 blocks (718 SVs, 389 regions) ...Zebrafish v. chicken: 6558 hits (4154 anchors) in 573 blocks (434 SVs, 371 regions) ...human v. chicken: 15091 hits (11789 anchors) in 653 blocks (549 SVs, 394 regions) ...frog v. chicken: 14777 hits (10885 anchors) in 726 blocks (658 SVs, 316 regions) ...Zebrafish v. Lamprey: 1010 hits (442 anchors) in 99 blocks (42 SVs, 59 regions) ...frog v. Lamprey: 3678 hits (1474 anchors) in 317 blocks (191 SVs, 149 regions) ...human v. Lamprey: 3535 hits (1455 anchors) in 304 blocks (157 SVs, 158 regions) ...Lamprey v. chicken: 3879 hits (1569 anchors) in 334 blocks (203 SVs, 144 regions) ...Zebrafish v. Amphioxus: 422 hits (56 anchors) in 10 blocks (2 SVs, 8 regions) ...frog v. Amphioxus: 591 hits (138 anchors) in 30 blocks (9 SVs, 21 regions) ...human v. Amphioxus: 764 hits (187 anchors) in 35 blocks (11 SVs, 24 regions) ...chicken v. Amphioxus: 709 hits (185 anchors) in 42 blocks (19 SVs, 23 regions) ...Lamprey v. Amphioxus: 782 hits (304 anchors) in 69 blocks (33 SVs, 36 regions) ...Amphioxus v. Amphioxus: 27337 hits (17491 anchors) in 297 blocks (0 SVs, 0 regions)

############################

5. Building synteny-constrained orthogroups ... ############## Running Orthofinder within syntenic regions ...Zebrafish v. Zebrafish: <50% (22.8%) of syn. hits in regions with >=40 genes, not running ...frog v. frog: <50% (34.7%) of syn. hits in regions with >=40 genes, not running ...human v. human: <50% (19.6%) of syn. hits in regions with >=40 genes, not running ...chicken v. chicken: n syn / syn OG / inblk OG hits = 60833 / 52092 / 52662 ...Lamprey v. Lamprey: <50% (29.5%) of syn. hits in regions with >=40 genes, not running ...Zebrafish v. frog: <50% (12.5%) of syn. hits in regions with >=40 genes, not running ...Zebrafish v. human: <50% (13.3%) of syn. hits in regions with >=40 genes, not running ...frog v. human: n syn / syn OG / inblk OG hits = 15139 / 12270 / 12653 ...Zebrafish v. chicken: <50% (11.2%) of syn. hits in regions with >=40 genes, not running ...human v. chicken: n syn / syn OG / inblk OG hits = 15091 / 12565 / 12812 ...frog v. chicken: n syn / syn OG / inblk OG hits = 14777 / 12030 / 12397 ...Zebrafish v. Lamprey: <50% (0%) of syn. hits in regions with >=40 genes, not running ...frog v. Lamprey: <50% (18.2%) of syn. hits in regions with >=40 genes, not running ...human v. Lamprey: <50% (11.6%) of syn. hits in regions with >=40 genes, not running ...Lamprey v. chicken: <50% (30.7%) of syn. hits in regions with >=40 genes, not running ...Zebrafish v. Amphioxus: <50% (0%) of syn. hits in regions with >=40 genes, not running ...frog v. Amphioxus: <50% (0%) of syn. hits in regions with >=40 genes, not running ...human v. Amphioxus: <50% (0%) of syn. hits in regions with >=40 genes, not running ...chicken v. Amphioxus: <50% (0%) of syn. hits in regions with >=40 genes, not running ...Lamprey v. Amphioxus: <50% (0%) of syn. hits in regions with >=40 genes, not running ...Amphioxus v. Amphioxus: no non-self blocks ############## Pulling syntenic orthogroups Done!

############################

6. Integrating syntenic positions across genomes ... ############## Generating syntenic dotplots ... Done! ############## Interpolating syntenic positions of genes ... human: (0 / 1 / 2 / >2 syntenic positions) Amphioxus: 12770 / 1989 / 5 / 30 Lamprey : 9120 / 4727 / 1006 / 134 Zebrafish: 13021 / 7776 / 805 / 25 chicken : 1665 / 12564 / 1092 / 56 frog : 2844 / 12649 / 1579 / 67 human : 0 / 12925 / 5947 / 1806 frog: (0 / 1 / 2 / >2 syntenic positions) Amphioxus: 13322 / 1393 / 51 / 27 Lamprey : 9714 / 4033 / 1150 / 90 Zebrafish: 13842 / 7312 / 458 / 19 chicken : 2326 / 11991 / 985 / 54 frog : 0 / 12692 / 7200 / 2006 human : 3555 / 12650 / 1196 / 57 Zebrafish: (0 / 1 / 2 / >2 syntenic positions) Amphioxus: 14183 / 573 / 6 / 29 Lamprey : 12935 / 1839 / 161 / 43 Zebrafish: 0 / 20950 / 5162 / 393 chicken : 7308 / 6019 / 1849 / 119 frog : 8513 / 6756 / 1688 / 110 human : 8483 / 6687 / 2077 / 147 chicken: (0 / 1 / 2 / >2 syntenic positions) Amphioxus: 13265 / 1221 / 282 / 26 Lamprey : 9304 / 4491 / 1028 / 163 Zebrafish: 13542 / 7710 / 362 / 17 chicken : 0 / 9411 / 6097 / 2581 frog : 3316 / 12745 / 1030 / 48 human : 3091 / 13190 / 1153 / 46 Lamprey: (0 / 1 / 2 / >2 syntenic positions) Amphioxus: 10576 / 3858 / 327 / 30 Lamprey : 0 / 13051 / 3959 / 557 Zebrafish: 18748 / 2613 / 230 / 0 chicken : 8562 / 4806 / 1641 / 237 frog : 9568 / 5750 / 1563 / 143 human : 10469 / 5511 / 1241 / 142 Amphioxus: (0 / 1 / 2 / >2 syntenic positions) Amphioxus: 1 / 17519 / 0 / 0 Lamprey : 12769 / 2128 / 79 / 0 Zebrafish: 21091 / 502 / 0 / 0 chicken : 13775 / 1210 / 253 / 0 frog : 15915 / 1095 / 2 / 0 human : 16062 / 1215 / 71 / 0 Done!

############################

7. Final block coordinate calculation and riparian plotting ... ############## Calculating syntenic blocks by reference chromosomes ... n regions (aggregated by 25 gene radius): 6511 n blocks (collinear sets of > 5 genes): 9100 ############## Building ref.-phased blks and riparian plots for haploid genomes: Error in x$dist[[i]] : subscript out of bounds In addition: There were 50 or more warnings (use warnings() to see the first 50)

   warnings() Warning messages: 1: In system2(ofcall, comm, stdout = TRUE, stderr = TRUE) : running command ''orthofinder' -b /Users/tommysalvetti/desktop/genomeRepo/tmp/reg21/orthofinder/Results_May10/WorkingDirectory -t 1 -a 1 -X 2>&1' had status 1 2: In system2(ofcall, comm, stdout = TRUE, stderr = TRUE) : running command ''orthofinder' -b /Users/tommysalvetti/desktop/genomeRepo/tmp/reg22/orthofinder/Results_May10/WorkingDirectory -t 1 -a 1 -X 2>&1' had status 1 3: In system2(ofcall, comm, stdout = TRUE, stderr = TRUE) : running command ''orthofinder' -b /Users/tommysalvetti/desktop/genomeRepo/tmp/reg67/orthofinder/Results_May10/WorkingDirectory -t 1 -a 1 -X 2>&1' had status 1 4: In system2(ofcall, comm, stdout = TRUE, stderr = TRUE) : running command ''orthofinder' -b /Users/tommysalvetti/desktop/genomeRepo/tmp/reg95/orthofinder/Results_May10/WorkingDirectory -t 1 -a 1 -X 2>&1' had status 1 5: In system2(ofcall, comm, stdout = TRUE, stderr = TRUE) : running command ''orthofinder' -b /Users/tommysalvetti/desktop/genomeRepo/tmp/reg28/orthofinder/Results_May10/WorkingDirectory -t 1 -a 1 -X 2>&1' had status 1 6: In system2(ofcall, comm, stdout = TRUE, stderr = TRUE) : running command ''orthofinder' -b /Users/tommysalvetti/desktop/genomeRepo/tmp/reg31/orthofinder/Results_May10/WorkingDirectory -t 1 -a 1 -X 2>&1' had status 1 7: In system2(ofcall, comm, stdout = TRUE, stderr = TRUE) : running command ''orthofinder' -b /Users/tommysalvetti/desktop/genomeRepo/tmp/reg32/orthofinder/Results_May10/WorkingDirectory -t 1 -a 1 -X 2>&1' had status 1 8: In system2(ofcall, comm, stdout = TRUE, stderr = TRUE) : running command ''orthofinder' -b /Users/tommysalvetti/desktop/genomeRepo/tmp/reg39/orthofinder/Results_May10/WorkingDirectory -t 1 -a 1 -X 2>&1' had status 1 9: In system2(ofcall, comm, stdout = TRUE, stderr = TRUE) : running command ''orthofinder' -b /Users/tommysalvetti/desktop/genomeRepo/tmp/reg40/orthofinder/Results_May10/WorkingDirectory -t 1 -a 1 -X 2>&1' had status 1 10: In system2(ofcall, comm, stdout = TRUE, stderr = TRUE) : running command ''orthofinder' -b /Users/tommysalvetti/desktop/genomeRepo/tmp/reg6/orthofinder/Results_May10/WorkingDirectory -t 1 -a 1 -X 2>&1' had status 1 11: In system2(ofcall, comm, stdout = TRUE, stderr = TRUE) : running command ''orthofinder' -b /Users/tommysalvetti/desktop/genomeRepo/tmp/reg44/orthofinder/Results_May10/WorkingDirectory -t 1 -a 1 -X 2>&1' had status 1 12: In system2(ofcall, comm, stdout = TRUE, stderr = TRUE) : running command ''orthofinder' -b /Users/tommysalvetti/desktop/genomeRepo/tmp/reg46/orthofinder/Results_May10/WorkingDirectory -t 1 -a 1 -X 2>&1' had status 1 13: In system2(ofcall, comm, stdout = TRUE, stderr = TRUE) : running command ''orthofinder' -b /Users/tommysalvetti/desktop/genomeRepo/tmp/reg49/orthofinder/Results_May10/WorkingDirectory -t 1 -a 1 -X 2>&1' had status 1 14: In system2(ofcall, comm, stdout = TRUE, stderr = TRUE) : running command ''orthofinder' -b /Users/tommysalvetti/desktop/genomeRepo/tmp/reg23/orthofinder/Results_May10/WorkingDirectory -t 1 -a 1 -X 2>&1' had status 1 15: In system2(ofcall, comm, stdout = TRUE, stderr = TRUE) : running command ''orthofinder' -b /Users/tommysalvetti/desktop/genomeRepo/tmp/reg60/orthofinder/Results_May10/WorkingDirectory -t 1 -a 1 -X 2>&1' had status 1 16: In system2(ofcall, comm, stdout = TRUE, stderr = TRUE) : running command ''orthofinder' -b /Users/tommysalvetti/desktop/genomeRepo/tmp/reg63/orthofinder/Results_May10/WorkingDirectory -t 1 -a 1 -X 2>&1' had status 1 17: In system2(ofcall, comm, stdout = TRUE, stderr = TRUE) : running command ''orthofinder' -b /Users/tommysalvetti/desktop/genomeRepo/tmp/reg69/orthofinder/Results_May10/WorkingDirectory -t 1 -a 1 -X 2>&1' had status 1 18: In system2(ofcall, comm, stdout = TRUE, stderr = TRUE) : running command ''orthofinder' -b /Users/tommysalvetti/desktop/genomeRepo/tmp/reg70/orthofinder/Results_May10/WorkingDirectory -t 1 -a 1 -X 2>&1' had status 1 19: In system2(ofcall, comm, stdout = TRUE, stderr = TRUE) : running command ''orthofinder' -b /Users/tommysalvetti/desktop/genomeRepo/tmp/reg73/orthofinder/Results_May10/WorkingDirectory -t 1 -a 1 -X 2>&1' had status 1 20: In system2(ofcall, comm, stdout = TRUE, stderr = TRUE) : running command ''orthofinder' -b /Users/tommysalvetti/desktop/genomeRepo/tmp/reg13/orthofinder/Results_May10/WorkingDirectory -t 1 -a 1 -X 2>&1' had status 1 21: In system2(ofcall, comm, stdout = TRUE, stderr = TRUE) : running command ''orthofinder' -b /Users/tommysalvetti/desktop/genomeRepo/tmp/reg14/orthofinder/Results_May10/WorkingDirectory -t 1 -a 1 -X 2>&1' had status 1 22: In system2(ofcall, comm, stdout = TRUE, stderr = TRUE) : running command ''orthofinder' -b /Users/tommysalvetti/desktop/genomeRepo/tmp/reg16/orthofinder/Results_May10/WorkingDirectory -t 1 -a 1 -X 2>&1' had status 1 23: In system2(ofcall, comm, stdout = TRUE, stderr = TRUE) : running command ''orthofinder' -b /Users/tommysalvetti/desktop/genomeRepo/tmp/reg75/orthofinder/Results_May10/WorkingDirectory -t 1 -a 1 -X 2>&1' had status 1 24: In system2(ofcall, comm, stdout = TRUE, stderr = TRUE) : running command ''orthofinder' -b /Users/tommysalvetti/desktop/genomeRepo/tmp/reg82/orthofinder/Results_May10/WorkingDirectory -t 1 -a 1 -X 2>&1' had status 1 25: In system2(ofcall, comm, stdout = TRUE, stderr = TRUE) : running command ''orthofinder' -b /Users/tommysalvetti/desktop/genomeRepo/tmp/reg86/orthofinder/Results_May10/WorkingDirectory -t 1 -a 1 -X 2>&1' had status 1 26: In system2(ofcall, comm, stdout = TRUE, stderr = TRUE) : running command ''orthofinder' -b /Users/tommysalvetti/desktop/genomeRepo/tmp/reg88/orthofinder/Results_May10/WorkingDirectory -t 1 -a 1 -X 2>&1' had status 1 27: In system2(ofcall, comm, stdout = TRUE, stderr = TRUE) : running command ''orthofinder' -b /Users/tommysalvetti/desktop/genomeRepo/tmp/reg102/orthofinder/Results_May10/WorkingDirectory -t 1 -a 1 -X 2>&1' had status 1 28: In system2(ofcall, comm, stdout = TRUE, stderr = TRUE) : running command ''orthofinder' -b /Users/tommysalvetti/desktop/genomeRepo/tmp/reg128/orthofinder/Results_May10/WorkingDirectory -t 1 -a 1 -X 2>&1' had status 1 29: In system2(ofcall, comm, stdout = TRUE, stderr = TRUE) : running command ''orthofinder' -b /Users/tommysalvetti/desktop/genomeRepo/tmp/reg131/orthofinder/Results_May10/WorkingDirectory -t 1 -a 1 -X 2>&1' had status 1 30: In system2(ofcall, comm, stdout = TRUE, stderr = TRUE) : running command ''orthofinder' -b /Users/tommysalvetti/desktop/genomeRepo/tmp/reg141/orthofinder/Results_May10/WorkingDirectory -t 1 -a 1 -X 2>&1' had status 1 31: In system2(ofcall, comm, stdout = TRUE, stderr = TRUE) : running command ''orthofinder' -b /Users/tommysalvetti/desktop/genomeRepo/tmp/reg162/orthofinder/Results_May10/WorkingDirectory -t 1 -a 1 -X 2>&1' had status 1 32: In system2(ofcall, comm, stdout = TRUE, stderr = TRUE) : running command ''orthofinder' -b /Users/tommysalvetti/desktop/genomeRepo/tmp/reg176/orthofinder/Results_May10/WorkingDirectory -t 1 -a 1 -X 2>&1' had status 1 33: In system2(ofcall, comm, stdout = TRUE, stderr = TRUE) : running command ''orthofinder' -b /Users/tommysalvetti/desktop/genomeRepo/tmp/reg237/orthofinder/Results_May10/WorkingDirectory -t 1 -a 1 -X 2>&1' had status 1 34: In system2(ofcall, comm, stdout = TRUE, stderr = TRUE) : running command ''orthofinder' -b /Users/tommysalvetti/desktop/genomeRepo/tmp/reg255/orthofinder/Results_May10/WorkingDirectory -t 1 -a 1 -X 2>&1' had status 1 35: In system2(ofcall, comm, stdout = TRUE, stderr = TRUE) : running command ''orthofinder' -b /Users/tommysalvetti/desktop/genomeRepo/tmp/reg262/orthofinder/Results_May10/WorkingDirectory -t 1 -a 1 -X 2>&1' had status 1 36: In system2(ofcall, comm, stdout = TRUE, stderr = TRUE) : running command ''orthofinder' -b /Users/tommysalvetti/desktop/genomeRepo/tmp/reg272/orthofinder/Results_May10/WorkingDirectory -t 1 -a 1 -X 2>&1' had status 1 37: In system2(ofcall, comm, stdout = TRUE, stderr = TRUE) : running command ''orthofinder' -b /Users/tommysalvetti/desktop/genomeRepo/tmp/reg291/orthofinder/Results_May10/WorkingDirectory -t 1 -a 1 -X 2>&1' had status 1 38: In system2(ofcall, comm, stdout = TRUE, stderr = TRUE) : running command ''orthofinder' -b /Users/tommysalvetti/desktop/genomeRepo/tmp/reg289/orthofinder/Results_May10/WorkingDirectory -t 1 -a 1 -X 2>&1' had status 1 39: In system2(ofcall, comm, stdout = TRUE, stderr = TRUE) : running command ''orthofinder' -b /Users/tommysalvetti/desktop/genomeRepo/tmp/reg328/orthofinder/Results_May10/WorkingDirectory -t 1 -a 1 -X 2>&1' had status 1 40: In system2(ofcall, comm, stdout = TRUE, stderr = TRUE) : running command ''orthofinder' -b /Users/tommysalvetti/desktop/genomeRepo/tmp/reg331/orthofinder/Results_May10/WorkingDirectory -t 1 -a 1 -X 2>&1' had status 1 41: In system2(ofcall, comm, stdout = TRUE, stderr = TRUE) : running command ''orthofinder' -b /Users/tommysalvetti/desktop/genomeRepo/tmp/reg339/orthofinder/Results_May10/WorkingDirectory -t 1 -a 1 -X 2>&1' had status 1 42: In system2(ofcall, comm, stdout = TRUE, stderr = TRUE) : running command ''orthofinder' -b /Users/tommysalvetti/desktop/genomeRepo/tmp/reg360/orthofinder/Results_May10/WorkingDirectory -t 1 -a 1 -X 2>&1' had status 1 43: In system2(ofcall, comm, stdout = TRUE, stderr = TRUE) : running command ''orthofinder' -b /Users/tommysalvetti/desktop/genomeRepo/tmp/reg361/orthofinder/Results_May10/WorkingDirectory -t 1 -a 1 -X 2>&1' had status 1 44: In system2(ofcall, comm, stdout = TRUE, stderr = TRUE) : running command ''orthofinder' -b /Users/tommysalvetti/desktop/genomeRepo/tmp/reg381/orthofinder/Results_May10/WorkingDirectory -t 1 -a 1 -X 2>&1' had status 1 45: In system2(ofcall, comm, stdout = TRUE, stderr = TRUE) : running command ''orthofinder' -b /Users/tommysalvetti/desktop/genomeRepo/tmp/reg384/orthofinder/Results_May10/WorkingDirectory -t 1 -a 1 -X 2>&1' had status 1 46: In system2(ofcall, comm, stdout = TRUE, stderr = TRUE) : running command ''orthofinder' -b /Users/tommysalvetti/desktop/genomeRepo/tmp/reg14/orthofinder/Results_May10/WorkingDirectory -t 1 -a 1 -X 2>&1' had status 1 47: In system2(ofcall, comm, stdout = TRUE, stderr = TRUE) : running command ''orthofinder' -b /Users/tommysalvetti/desktop/genomeRepo/tmp/reg20/orthofinder/Results_May10/WorkingDirectory -t 1 -a 1 -X 2>&1' had status 1 48: In system2(ofcall, comm, stdout = TRUE, stderr = TRUE) : running command ''orthofinder' -b /Users/tommysalvetti/desktop/genomeRepo/tmp/reg34/orthofinder/Results_May10/WorkingDirectory -t 1 -a 1 -X 2>&1' had status 1 49: In system2(ofcall, comm, stdout = TRUE, stderr = TRUE) : running command ''orthofinder' -b /Users/tommysalvetti/desktop/genomeRepo/tmp/reg37/orthofinder/Results_May10/WorkingDirectory -t 1 -a 1 -X 2>&1' had status 1 50: In system2(ofcall, comm, stdout = TRUE, stderr = TRUE) : running command ''orthofinder' -b /Users/tommysalvetti/desktop/genomeRepo/tmp/reg42/orthofinder/Results_May10/WorkingDirectory -t 1 -a 1 -X 2>&1' had status 1

[jtlovell]

This error can occur if you do not have a proper orthofinder installation, but set orthofinderInBlk = TRUE, which is the default if any ploidy > 1. If you are sure you have a good orthofinder install (any can call orthofinder -h successfully from R), then I'm not sure. That said, I would really suggest not using GENESPACE for this ... there is basically no synteny between two or more of your genomes.