Hi everyone,

so i am trying to extract the 1000bp upstream, for every gene in solanum pennellii, i know i would need to build the txdb object and the bsgenome for this, firstly i downloaded the fasta format genome and the gff file from ncbi genome database

http://www.ncbi.nlm.nih.gov/genome/?term=solanum+pennellii

after that , i used this perl script to split the genome of s.pennellii into chromosomes and build the bsgenome from these 12 chromosomes.

#!/usr/bin/perl

$f = $ARGV[0]; #get the file name

open (INFILE, "<$f")
or die "Can't open: $f $!";

while (<INFILE>) {
$line = $_;
chomp $line;
if ($line =~ /\>/) { #if has fasta >
close OUTFILE;
$new_file = substr($line,1);
$new_file .= ".fa";
open (OUTFILE, ">$new_file")
or die "Can't open: $new_file $!";
}
print OUTFILE "$line\n";
}
close OUTFILE;

__________________________________________

then i created the seed file and forge the bsgenome package as the instructions. there are actually no errors when checking the built package. 

Package:BSgenome.Slycopersicum.ensembl.Heinz1706
Title:Full genome sequences for Solanum lycopersicum
Description:Full genome sequences for Solanmum lycopersicum provided by ensembl
Version:1.0
organism:Solanum lycopersicum
common_name:Tomato
provider:ensembl
provider_version:Heinz1706
release_date:Jan.2016
release_name:S.lyco Genome Sequencing
source_url:ftp://ftp.ensemblgenomes.org/pub/plants/release-30/fasta/solanum_lycopersicum/dna/
organism_biocview: Solanum_lycopersicum
BSgenomeObjname: Slycopersicum
seqnames: paste("chr", c(1:12), sep="")
circ_seqs: NULL
mseqnames: NULL
SrcDataFiles:ftp://ftp.ensemblgenomes.org/pub/plants/release-30/fasta/solanum_lycopersicum/dna/ PkgExamples: genome$GL896898 # same as genome[["GL896898"]] 
seqs_srcdir: ~/Desktop/S.lyco

_________________________

Unfortunately, when i typed the code, the test return something weird , also the seqinfo returns an error too

library("BSgenome.Spennellii.ncbi.SPENNV200")
test<-library("BSgenome.Spennellii.ncbi.SPENNV200")
> test
 [1] "BSgenome.Athaliana.TAIR.TAIR9"      
 [2] "TxDb.Athaliana.BioMart.plantsmart28"
 [3] "GenomicFeatures"                    
 [4] "AnnotationDbi"                      
 [5] "Biobase"                            
 [6] "BSgenome.Spennellii.ncbi.SPENNV200" 
 [7] "BSgenome"                           
 [8] "rtracklayer"                        
 [9] "Biostrings"                         
[10] "XVector"                            
[11] "GenomicRanges"                      
[12] "GenomeInfoDb"                       
[13] "IRanges"                            
[14] "S4Vectors"                          
[15] "stats4"                             
[16] "BiocGenerics"                       
[17] "parallel"                           
[18] "stats"                              
[19] "graphics"                           
[20] "grDevices"                          
[21] "utils"                              
[22] "datasets"                           
[23] "methods"                            
[24] "base" 

> seqinfo(test)
Error in (function (classes, fdef, mtable)  : 
  unable to find an inherited method for function ‘seqinfo’ for signature ‘"character"’

_______________________

for the txdb object , the code i used was as below and it looks wrong to me as well.

gtffile <- file.path("~/Desktop/s.pen/gff/GCF_001406875.1_SPENNV200_genomic.gff")
makeTxDbFromGFF(gtffile)
txdb <- makeTxDbFromGFF(gtffile)

seqinfo(txdb)

Seqinfo object with 12 sequences from an unspecified genome; no seqlengths:
  seqnames    seqlengths isCircular genome
  NC_028637.1       <NA>       <NA>   <NA>
  NC_028638.1       <NA>       <NA>   <NA>
  NC_028639.1       <NA>       <NA>   <NA>
  NC_028640.1       <NA>       <NA>   <NA>
  NC_028641.1       <NA>       <NA>   <NA>
  ...                ...        ...    ...
  NC_028644.1       <NA>       <NA>   <NA>
  NC_028645.1       <NA>       <NA>   <NA>
  NC_028646.1       <NA>       <NA>   <NA>
  NC_028647.1       <NA>       <NA>   <NA>
  NC_028648.1       <NA>       <NA>   <NA>

can anybody tell me may be i have done something wrong?? thank you very much.

> sessionInfo()

R version 3.2.3 (2015-12-10)
Platform: x86_64-pc-linux-gnu (64-bit)
Running under: Ubuntu 14.04.3 LTS

locale:
 [1] LC_CTYPE=en_GB.UTF-8       LC_NUMERIC=C              
 [3] LC_TIME=en_GB.UTF-8        LC_COLLATE=en_GB.UTF-8    
 [5] LC_MONETARY=en_GB.UTF-8    LC_MESSAGES=en_GB.UTF-8   
 [7] LC_PAPER=en_GB.UTF-8       LC_NAME=C                 
 [9] LC_ADDRESS=C               LC_TELEPHONE=C            
[11] LC_MEASUREMENT=en_GB.UTF-8 LC_IDENTIFICATION=C       

attached base packages:
[1] stats4    parallel  stats     graphics  grDevices utils     datasets 
[8] methods   base     

other attached packages:
 [1] BSgenome.Athaliana.TAIR.TAIR9_1.3.1000   
 [2] TxDb.Athaliana.BioMart.plantsmart28_3.2.2
 [3] GenomicFeatures_1.22.12                  
 [4] AnnotationDbi_1.32.3                     
 [5] Biobase_2.30.0                           
 [6] BSgenome.Spennellii.ncbi.SPENNV200_1.0   
 [7] BSgenome_1.38.0                          
 [8] rtracklayer_1.30.1                       
 [9] Biostrings_2.38.3                        
[10] XVector_0.10.0                           
[11] GenomicRanges_1.22.4                     
[12] GenomeInfoDb_1.6.3                       
[13] IRanges_2.4.6                            
[14] S4Vectors_0.8.11                         
[15] BiocGenerics_0.16.1                      

loaded via a namespace (and not attached):
 [1] zlibbioc_1.16.0            GenomicAlignments_1.6.3   
 [3] BiocParallel_1.4.3         tools_3.2.3               
 [5] SummarizedExperiment_1.0.2 DBI_0.3.1                 
 [7] lambda.r_1.1.7             futile.logger_1.4.1       
 [9] futile.options_1.0.0       bitops_1.0-6              
[11] biomaRt_2.26.1             RCurl_1.95-4.7            
[13] RSQLite_1.0.0              Rsamtools_1.22.0          
[15] XML_3.98-1.3    

An alternative approach is to load relevant libraries

library(GenomicFeatures); library(rtracklayer); library(BSgenome)

Make the TxDb from the GFF

txdb = makeTxDbFromGFF("GCF_001406875.1_SPENNV200_genomic.gff.gz")

Import the fasta file, and export as an (indexed) 2bit file, and reference the 2bit file

fa = FastaFile("GCF_001406875.1_SPENNV200_genomic.fna.gz")
export(import(fa), "GCF_001406875.1_SPENNV200_genomic.2bit")
twob = TwoBitFile("GCF_001406875.1_SPENNV200_genomic.2bit")

For rtracklayer versions before 1.31.4, the names in the fasta file need to be cleaned

dna = import(fa)
names(dna) = sapply(strsplit(names(dna), " "), "[[", 1)
export(dna, "GCF_001406875.1_SPENNV200_genomic.2bit")

You're then ready to go, e.g., 

> p = promoters(txdb)
> getSeq(twob, p)
  A DNAStringSet instance of length 43195
        width seq
    [1]  2200 AAATGCCAAAAAATTTTGTGGACGTCCGTTAA...CTCAAAACTTCAGGTAATTAATTATTATTAT
    [2]  2200 CCAAAGTAAAAATTGATTGAAAAATATTAAAA...GAACAAAAAGAATGAAGAAACGAAGTTACTA
    [3]  2200 CAAAGTAAAAATTGATTGAAAAATATTAAAAC...AACAAAAAGAATGAAGAAACGAAGTTACTAA
    [4]  2200 AAAGTAAAAATTGATTGAAAAATATTAAAACA...ACAAAAAGAATGAAGAAACGAAGTTACTAAG
    [5]  2200 AAAGTAAAAATTGATTGAAAAATATTAAAACA...ACAAAAAGAATGAAGAAACGAAGTTACTAAG
    ...   ... ...
[43191]  2200 TATGAGATCGTAAATAAGATAGGAAGAGGAGC...TATGATACACCTGTGCCAAGTTGGCGCATTT
[43192]  2200 GAAAGAGCTAGCTGAGTAGTGTACCCAATAAT...TTGATTTTCAAGCAAAAAACTTAGGAAAAAT
[43193]  2200 CGTTTGAATTCTTCAACAAGTTTTCAACAGCG...TTGATGGGACCTCGAAGAAGAGGGTCTGGTC
[43194]  2200 TTCCAATTAAGTTTAAAAATAAAACGTTGAAG...GCTTAAATGGGTAAGTCTTGAACCACTTTCC
[43195]  2200 AAGCAAAATTAATCAATAAAAAACAAAGCAGA...ACTTCATCTCAACTCACGAAGAGTGTCCACC

See also getPromoterSeq().