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I am trying to do a synteny analysis using MCScanX. As per requirement I have done all by all blast between two sets of protein from two closely related species. And also prepared the gff file. The file formats are as follows:

cyp_dan.blast

NP_001347641.1  XP_042591522.1  78.31   166     32      1       1       166     1       162     5e-91   267
NP_001347641.1  XP_042577882.1  60.24   166     62      2       1       166     1       162     4e-63   196
.
.
XP_704272.2     XP_018953536.1  31.03   87      59      1       275     360     90      176     8e-07   50.4
XP_704272.2     NP_001005958.2  33.33   63      38      1       273     331     38      100     1e-06   48.9
XP_704272.2     XP_018969808.1  31.33   83      56      1       275     356     91      173     2e-06   49.3```

cyp_dan.gff
NC_056572.1     gene-LOC1221465.1       2966    7093
NC_056572.1     id-LOC122146575.1       2966    3166
NC_056572.1     id-LOC122146575.1       3246    3357
.
.
NC_002333.2     gene-trnP;Dbxre.1       16527   16596
NC_002333.2     rna-trnP;Parent.1       16527   16596
NC_002333.2     exon-trnP-1;Par.1       16527   16596

cmdline: /home/software/MCScanX/MCScanX cyp_dan
Output:
############### Parameters ###############
# MATCH_SCORE: 50
# MATCH_SIZE: 5
# GAP_PENALTY: -1
# OVERLAP_WINDOW: 5
# E_VALUE: 1e-05
# MAX GAPS: 25
############### Statistics ###############
# Number of collinear genes: 0, Percentage: 0.00
# Number of all genes: 108556
##########################################

Here, both of the species are from same family since there is no chance to have "zero collinear gene".

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I'm not familiar with the MCScanX tool, but I see two potential issues that are worth investigating.

  1. The cyp_dan.gff file does not look like GFF or any of its common variants (GFF3, GTF, etc.). Again, I'm unfamiliar with MCScanX, so I don't know precisely how its wants the input data formatted. But if it wants a GFF file, I would double-check to make sure that what you have matches what the tools expects/requires.

  2. The gene/protein IDs in the cyp_dan.gff file do not seem to match the query or subject IDs in the cyp_dan.blast file. No tool is going to be able to find collinear adjacent genes if the IDs in the BLAST results don't match the IDs in the gene annotations.

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  • $\begingroup$ Hi, Thanks for your response. 1. Original gff format is not allowed to use. It requires modification with having chr name, geneID, start and end position only. I took out the necessary info using following cmd: ` awk -F "\t" '{a=substr($9,4,15)}$0~/gene/{print $1 "\t" a".1" "\t" $4 "\t" $5}' A.gff > A_new.gff ` 2. I have extracted the info from the NCBI annotated file and didnt make any change in ID info by my self. And after extracting this is how it looks. Can you please give me any suggestion to find exact match between blast and gff file? $\endgroup$ Apr 6, 2022 at 9:47

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