Problem: ABACAS doesn't design primer

[illnoobina]

Hi to everybody,

I am in the process of finishing my Contigs to a complete sequence and try to use ABACAS to assist me in gap closure. I want ABACAS to take over the primer design for me but it refuses to put out primer sequences. The tool runs as expected, MUMMER and Primer3 are installed and should be in $PATH. ABACAS creates the primer files (which are empty) and doesn't report errors in the primer design problem:

"Please wait... extracting target regions ...
26 gaps found in target sequence
Please wait...
Looking for primers...
Running Primer3 and checking uniquness of primers...
Counting left and right primer hits from a nucmer mapping (-c 15 -l 15)

PRIMER DESIGN DONE"

On Biostars someone had similar problems but nobody posted a solution to it. Did someone had similar issues and found a solution? Is something wrong with my Primer3?

Also: Everything else seems to work fine. I get reference maps and can view annotated version in ACT.

Thanks,

[illnoobina]

Okay so I did some more research and found that this problem is happening more often than expected. My deep googling took me to a message board where someone claims that it is a known issue for some genomes and there is no solution for it, sadly.

So I went on and I used the Projector2 webinterface that seemed to do quite well. Yet the ouptut I felt is somewhat not optimal, especially if you want to validate your output before ordering some dozens of oligos. I took the primer list it created and made a .fasta file by hand (name of primer + sequence). Took me 5 minutes to do.

Then I took my Contigs and used command line blast to blast all primers against my Contigs:

blastn -query /contigs.fasta -task blastn-short -out /PrimerBlast.txt -subject /Primer.fasta

(just replace "/contigs.fasta", "/PrimerBlast.txt","/Primer.fasta")

The output is obviously messy and I put it into order using a small R Script (note: my Primer length is 20, for different a length it needs to be adjusted):

ReadPrimerBlast <- readLines("/PrimerBlast.txt")

blastlist<-c()

for(i in c(1:length(ReadPrimerBlast))){
  if(substr(ReadPrimerBlast[i],1,6) == "Query=" && substr(ReadPrimerBlast[i+10],1,19) == " Identities = 20/20"){
    blastlist<-rbind(blastlist,c(ReadPrimerBlast[i],ReadPrimerBlast[i+4],ReadPrimerBlast[i+2],ReadPrimerBlast[i+13]))
  }    
}

My output looks like that (just the first 3 lines):
[1,] "Query= NODE_1_length_47713_cov_19.718735" "Subject= assembly_gap33_R" "Length=47863" "Query 447 GAGATCAACTGCTCATGCCA 466"
[2,] "Query= NODE_2_length_117362_cov_18.628407" "Subject= assembly_gap32_R" "Length=117512" "Query 116759 TCCTGCTGTGTGAACTGTCC 116778"
[3,] "Query= NODE_2_length_117362_cov_18.628407" "Subject= assembly_gap33_L" "Length=117512" "Query 726 CCCGATTGTACTCACCTCGT 745"

So now I can estimate where my primers bind from that and it looks quite good. Maybe this will help someone in the future.

If you have any comments don't hesitate to add them.

-illnoobina

edit: Projector2 linked

[jrvalverde]

Fixing ABACAS

ABACAS is designed to work with an earlier version of PRIMER3 which uses a different naming convention for the parameters. As a consequence, when ABACAS is run with a modern version of PRIMER3 it fails to recognize the old parameters, among them the sequence to search for primers, and since it sees no sequence, it ends with nothing done.

To fix it, the faster solution may be to use an old version of Primer3.

[illnoobina]

Hi jrvalverde,

Thanks for your reply. I had this suspicion too but I failed to get an earlier version of Primer 3 to work. I also tried the test files supplied with ABACAS which had similar problems. Furthermore, I tried many different Primer 3 parameters but all failed.

If I have time I might try to get an older Version of Primer 3 running. Right now I will stick to Projector2 since I already ordered the suggested Primers - I hope I can post my experiences using this finishing tool soon.

Best,

[touzain]

solution

Hello,

Yes, this is a problem of primer version (names of variables have changed).

In the abacas perl script, you only need to replace:

print FILE "PRIMER_SEQUENCE_ID=Starting_Pos $positions[$i]
SEQUENCE=$gappedSeq[$i]
PRIMER_OPT_SIZE=$opt
PRIMER_MIN_SIZE=$min
PRIMER_MAX_SIZE=$max
PRIMER_OPT_TM=$optTemp
PRIMER_MIN_TM=$minTemp
PRIMER_MAX_TM=$maxTemp
PRIMER_NUM_NS_ACCEPTED=1
PRIMER_PRODUCT_SIZE_RANGE=$lowRange-$maxRange
PRIMER_MIN_GC=$gcMin
PRIMER_GC_CLAMP =$gclamp
PRIMER_OPT_GC_PERCENT=$gcOpt
PRIMER_MAX_GC=$gcMax
PRIMER_INTERNAL_OLIGO_EXCLUDED_REGION=$exc1,$exclude $exc2,$exclude
".$qual."Number To Return=1
=\n";

by:

print FILE "P3_FILE_ID=title_you_want
SEQUENCE_ID=Starting_Pos $positions[$i]
SEQUENCE_TEMPLATE=$gappedSeq[$i]
PRIMER_OPT_SIZE=$opt
PRIMER_MIN_SIZE=$min
PRIMER_MAX_SIZE=$max
PRIMER_OPT_TM=$optTemp
PRIMER_MIN_TM=$minTemp
PRIMER_MAX_TM=$maxTemp
PRIMER_MAX_NS_ACCEPTED=1
PRIMER_PRODUCT_SIZE_RANGE=$lowRange-$maxRange
PRIMER_MIN_GC=$gcMin
PRIMER_GC_CLAMP =$gclamp
PRIMER_OPT_GC_PERCENT=$gcOpt
PRIMER_MAX_GC=$gcMax
SEQUENCE_INTERNAL_EXCLUDED_REGION=$exc1,$exclude $exc2,$exclude
PRIMER_NUM_RETURN=1
=\n";

Have a nice day
Fabrice