[rebecca_m_d]

Hi Boetsie,

I have been using SSPACE for scaffolding, and I would like to try your new software for gap filling. But first, I need help in understanding the final evidence from SSPACE. Can you please explain the example below? 1st line... Reverse of contig_93 has 17 links to forward of contig_17. But, is there an estimated gap of 164bp or an overlap of 164bp between contigs? The sequence region in question does not have lower case bases (indicating an overlap), but has only 1 lower case "n". Should I interpret all the lowercase n's as potential gaps? Have you been able to validate these gaps in the lab?

>scaffold16.1|size67863|tigs4
r_tig93|size18643|links17|gaps-164
f_tig17|size46195|links7|gaps-164
f_tig266|size1908|links15|gaps-169
r_tig282|size1114

Thanks in advance for your help!

[heath.obrien]

Hmmm, that is very strange. Any idea if the scaffolds contain very short sequences between gaps, e.g.;

AAGCTGCTAGNNNGTATGNNNNNAGGGTAGATAG

I think the script does not handle these patterns very well at the moment. I'm working on this.

Regards,
Boetsie

Most of the truncated sequences do not have short sequences between gaps, but they do have short sequences between the start of the scaffold and the first N. There also appears to be a relationship between the length of this sequence and the size of the truncated sequence.

[CPCantalapiedra]

Hey CPCantalarpiedra,

I'm having exactly the same problem. Can you elaborate on how you fixed your problem? Did you remove a symbolic link or put one in? Also in which directory is your bwa and bowtie installed? (i.e. /usr/local/bin?)

Thanks!

Megan

Umm maybe not the best way to do this, but I am working with the full path and the program in my local bin, so... ~/bin/GapFiller_v1-11_linux-x86_64/GapFiller.pl
bwa and bowtie are already in the same GapFiller folder. I hope this helps

[megancamilla]

GapFiller doesn't go through iterations

umm when pasting the bowtie line I realized that maybe the symbolik link was causing the problem, and it seems that it was the source of the problem in both cases.

Also, could you explain further when and what for using iterations? I have checked and the second iteration is closing gaps, so it seems useful. Why weren't closed during iteration1? It is just the discovery of previously unmapped reads, that now are able to completely map to one edge?

Thank you again!
CPC

Hey CPCantalarpiedra,

I'm having exactly the same problem. Can you elaborate on how you fixed your problem? Did you remove a symbolic link or put one in? Also in which directory is your bwa and bowtie installed? (i.e. /usr/local/bin?)

Thanks!

Megan

[boetsie]

Hi Martin,

I have been working with GapFiller and had great success with the first assembly that I tried it on, but I'm having a puzzling problem with my latest run: a large number of the contigs are being dramatically truncated. Before running GapFiller, the minimum contig size was 200 bp, but after running it there are over 1200 contigs shorter than 100 bp, with some as short as 2 bp. Do you (or anyone else) have any idea what might be going on here? I ran it with the default parameters.

Hmmm, that is very strange. Any idea if the scaffolds contain very short sequences between gaps, e.g.;

AAGCTGCTAGNNNGTATGNNNNNAGGGTAGATAG

I think the script does not handle these patterns very well at the moment. I'm working on this.

Regards,
Boetsie

[boetsie]

I've been trying to get GapFiller working on a number of Illumina assemblies that we have in my lab, but I'm not getting all of the output files that the manual says I should.

I ran the tutorial on the scaffolds in the example/ folder using the stipulated read sets, but I never get the following output files: XXX.filler.final.text, XXX.closed.evidence.txt, XXX.gapfilled.final.fa.

I do get the following files: XXX.summarfile.final.txt, XXX.gapclosure.fa and a bwa logfile in alignoutput/ and an empty XXX.closed.evidence.iteration1.txt file in intermediate_results/.

Has anyone else had this problem or have any suggestions for getting the correct output files?

Hi, somehow gapFiller stopped at a certain point. Can you look at the command line at see if there is any error?

Regards,Boetsie

[CPCantalapiedra]

I had the same problem sometimes. Don't know exactly why, but running again the analysis often works in my case, and the complete output file set is generated. You can try to run with several iterations and you can check in the output which iteration is stopping at. Maybe stderr could give some light too?

[mcnelson.phd]

I've been trying to get GapFiller working on a number of Illumina assemblies that we have in my lab, but I'm not getting all of the output files that the manual says I should.

I ran the tutorial on the scaffolds in the example/ folder using the stipulated read sets, but I never get the following output files: XXX.filler.final.text, XXX.closed.evidence.txt, XXX.gapfilled.final.fa.

I do get the following files: XXX.summarfile.final.txt, XXX.gapclosure.fa and a bwa logfile in alignoutput/ and an empty XXX.closed.evidence.iteration1.txt file in intermediate_results/.

Has anyone else had this problem or have any suggestions for getting the correct output files?

[heath.obrien]

Hi Martin,

I have been working with GapFiller and had great success with the first assembly that I tried it on, but I'm having a puzzling problem with my latest run: a large number of the contigs are being dramatically truncated. Before running GapFiller, the minimum contig size was 200 bp, but after running it there are over 1200 contigs shorter than 100 bp, with some as short as 2 bp. Do you (or anyone else) have any idea what might be going on here? I ran it with the default parameters.

[lizzyzhao]

errors in running

Hi Marten,

Thanks for the tool for gap filling. However, I had some problem running it , basically the problem is it stopped at the the bowtie-build which is the first step of bowtie. I checked the align output file, it turned out that asem1.contig.gpfill.gapclosure.fa is empty. I tried to read through your perl script but failed to understand how asem1.contig.gpfill.gapclosure.fa is generated so I couldn't figure out why this is empty. Could you please let me know the possible reason of it? Thank you very much!

-rw-r--r-- 1 users 40 2013-03-08 06:10 asem1.contig.gpfill.bowtieIndex.1.ebwt
-rw-r--r-- 1 users 4 2013-03-08 06:10 asem1.contig.gpfill.bowtieIndex.2.ebwt
-rw-r--r-- 1 users 0 2013-03-08 06:10 asem1.contig.gpfill.gapclosure.fa



perl ~/bin/GapFiller_v1-11_linux-x86_64/GapFiller.pl -l libraries -s asem1.contig -m 30 -o 3 -r 0.7 -n 10 -d 50 -t 0 -g 0 -T 1 -i 1 -b asem1.contig.gpfill

Your inserted inputs on [GapFiller_v1-11_Final] at Fri Mar 8 05:37:25 2013:
-s asem1.contig
-l libraries
-b asem1.contig.gpfill
-o 3
-m 30
-r 0.7
-n 10
-T 1
-g 0
-d 50
-t 0
-i 1


=>Fri Mar 8 05:37:25 2013: Reading and processing paired-read files

ITERATION 1:

=>Fri Mar 8 06:10:25 2013: Mapping reads to scaffolds, reading alignment output and storing reads
Warning: Empty input file
Reference file does not seem to be a FASTA file
Command: /home/bin/GapFiller_v1-11_linux-x86_64/bowtie/bowtie-build --quiet --noref asem1.contig.gpfill/alignoutput/asem1.contig.gpfill.gapclosure.fa asem1.contig.gpfill/alignoutput/asem1.contig.gpfill.bowtieIndex

Bowtie-build error; 256 at /home/bin/GapFiller_v1-11_linux-x86_64/GapFiller.pl line 242.

[boetsie]

Sorry for the late reply! Good that it solved the problem.

The iteration is indeed that previously unmapped reads are used for further closing the gap. This is especially useful if you used long insert size libraries for scaffolding.

Regards,
Boetsie

[CPCantalapiedra]

umm when pasting the bowtie line I realized that maybe the symbolik link was causing the problem, and it seems that it was the source of the problem in both cases.

Also, could you explain further when and what for using iterations? I have checked and the second iteration is closing gaps, so it seems useful. Why weren't closed during iteration1? It is just the discovery of previously unmapped reads, that now are able to completely map to one edge?

Thank you again!
CPC

[CPCantalapiedra]

Changing "bwa" to "bowtie" I got:

Bowtie-build error; -1 at ~/bin/gapfiller line 242.

[CPCantalapiedra]

Hi Boetsie,

I have used SSPACE with good success, and I congrat you for such a good software. Now I am trying GapFiller. I am using a single library with about 6M reads and a machine with 64 GB RAM. I use somewhat standard parameters, and have tried with -i 1 and -i 2, but the program stops after iteration1, without reporting any error. Just it stops after the "Mapping reads..." log. I am using "bwa" in library file, since my reads range from 36 up to 122 (I have the option to use only >100 bp reads if necessary).

I am wondering why this happens. Any idea?
Going to try the bowtie option too...

thanks
CPC

[boetsie]

Hi luisgls,

Set the -t option to 0, the -t will trim off by default 10 bases of your 'contig' edges, since we have seen that these are usually of bad quality.

Regards,
Boetsie

I have a finished genome assembled de novo (actually I also used SSPACE to identify maximal connections between final scaffolds). When I used GapFiller instead of reducing the number of Ns, it increased them. I compare the new N list with the original, and it correct some gaps, but sometimes in the original I had just 1 N, and now I have 7 Ns in this position. Can I turn off this sort of parameter?, why is it extending with 6 Ns?
Thanks