Maker Pipeline blast results

Question: Maker Pipeline blast results

5.2 years ago by

zgayk • 90

United States

zgayk • 90 wrote:

Hello,

I am hoping to use maker on a small cluster (~6 compute nodes) to annotate a fairly fragmented de novo assembly that has some longer contigs. We have maker installed, but so far even though every program runs, RepeatMasker seems to be the only program finding matches. Namely, blastx and exonerate don't find any alignment matches even though they seem to be set up correctly in the maker control file.

What I was wondering was whether this is an artifact of the fragmented assembly or some sort of setup error? I find the former hard to believe considering I got at least 2-3 blast hits for each longer contig in the entire assembly using galaxy megablast. I think the error lies in the fact that I get 0 hits, but I am not sure why:

Widget::blastx:

/usr/bin/blastx -db /tmp/maker_sHnU1b/chickenproteomeuniprot%2Efasta.mpi.10.9 -query /tmp/maker_sHnU1b/0/scaffold_1035.0 -num_alignments 10000 -num_descriptions 10000 -evalue 1e-06 -dbsize 300 -searchsp 500000000 -num_threads 1 -seg yes -soft_masking true -lcase_masking -show_gis -out /home/zgayk/MakerExample2/Gaviaimmerheader.maker.output/Gaviaimmerheader_datastore/38/7C/scaffold_1035//theVoid.scaffold_1035/0/scaffold_1035.0.chickenproteomeuniprot%2Efasta.blastx.temp_dir/chickenproteomeuniprot%2Efasta.mpi.10.9.blastx

#-------------------------------#

deleted:0 hits

collecting blastx reports

flattening protein clusters

prepare section files

processing the chunk divide

preparing evidence clusters for annotations

Preparing evidence for hint based annotation

clustering transcripts into genes for annotations

Processing transcripts into genes

choosing best annotation set

Choosing best annotations

processing chunk output

processing contig output

examining contents of the fasta file and run log

Essentially each .gff file produced for each contig is empty. If anyone knew how to fix this, I would be very appreciative.

Zach Gayk

annotation • 1.7k views

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modified 5.2 years ago by Juke34 • 4.5k • written 5.2 years ago by zgayk • 90

Could you tell us

- What is your N50 ?

- What did you fill for the min_contig parameter in the maker_opts.ctl ?

- What kind of proteins (database ?) do you try to align on your genome ?

- Which kind of genome do you try to annotate ? Bird ? Fungi ?

As specified in the "maker_opts.ctl", under 10kb try to annotate a sequence is often useless.

ADD REPLY • link written 5.2 years ago by Juke34 • 4.5k

5.2 years ago by

zgayk • 90

United States

zgayk • 90 wrote:

Hello, the assembly is fragmented:

Minimum    Number     Number     Total    Total    Scaffold
Scaffold   of     of     Scaffold     Contig     Contig
Length     Scaffolds    Contigs    Length     Length     Coverage
--------   --------------   --------------   --------------   --------------   --------
All    5,237,924   5,238,436   767,438,425   767,326,331   99.99%
50    3,616,441   3,616,953   710,236,525   710,124,431   99.98%
100    2,146,720   2,147,232   604,271,394   604,159,300   99.98%
250    743,885   744,397   394,016,485   393,904,391   99.97%
500    247,247   247,755   223,350,732   223,238,838   99.95%
1 KB    62,044   62,409   98,533,822   98,431,583   99.90%
2.5 KB    5,725   5,731   18,713,830   18,710,728   99.98%
5 KB    231   231   1,310,589   1,310,589   100.00%

The assembly is from a bird: the common loon (Gavia immer). I used the chicken (Gallus gallus) proteome as protein data, along with chicken cDNA for EST evidence. I put the minimum contig length at 500. The contig N50 is 814 bp.

Most of the assembly is in small contigs less than 1 kb, and I was only going to use maker as a trial. I thought it might be possible to get valid annotations for the longer contigs at least, but if you think this is not feasible let me know. The assembly was produced using abyss with pe read data and a k-mer size of 32. Then, because it was still so fragmented, I aligned the contigs to the available red-throated loon genome and this is what is shown. I am not sure why the assembly remain this fragmented (we have basically have no scaffolds), although it could be that the group that did the sequencing used one pe library (8kb). If there are any suggestions as to why the assembly remains so fragmented, I would be very interested. Are we too limited by having one insert library?

Thanks,

Zach

ADD COMMENT • link written 5.2 years ago by zgayk • 90

5.2 years ago by

Juke34 • 4.5k

Sweden

Juke34 • 4.5k wrote:

According to the size of your contigs, your Maker result it's not surprising. Moreover, the genes in that kind of genome are quite long.

I think you should focus your work on the assembly before to try to perform any annotation. You must improve significantly the size of your contigs ! I suggest you to try other assembly tools... but I'm not expert in this field.

good luck :)

ADD COMMENT • link modified 5.2 years ago • written 5.2 years ago by Juke34 • 4.5k

Does anyone have any ideas as to why the assembly is so fragmented, and specifically that no scaffolds are being produced? If I'm to improve the assembly I'll need to identify whether the current result is due to low quality data for making long contigs (only one insert size) or perhaps an error in the assembly process. I realize it is hard to determine from a distance, but any help would be appreciated.

Thanks,

Zach

ADD REPLY • link written 5.2 years ago by zgayk • 90

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