layout step produces empty gfa

[aechchiki]

Hi guys.

I am using minimap/miniasm to get the raw contigs by rapid assembly as preliminary step in order to perform error correction using @isovic 's racon.

My input file is: r7_2d.fastq, a file containing 2D MinION reads (RNA, not DNA sequencing).

Following your instructions, I installed latest minimap (Version: 0.2-r124-dirty) & miniasm (Version: 0.2-r137-dirty). Overlap step was completed succesfully (and got a non-empty paf.gz file), but output for layout (gfa file) is empty.

Here is my log:

# overlap: successful.
./minimap -Sw5 -L100 -m0 -t8 r7_2d.fastq r7_2d.fastq | gzip -1 > r7_2d.paf.gz
[M::mm_idx_gen::8.484*1.20] collected minimizers
[M::mm_idx_gen::9.471*1.72] sorted minimizers
[M::main::9.471*1.72] loaded/built the index for 113249 target sequence(s)
[M::main] max occurrences of a minimizer to consider: 37
[M::main] Version: 0.2-r124-dirty
[M::main] CMD: /home/aechchik/wgets/minimap/minimap -Sw5 -L100 -m0 -t8 r7_2d.fastq r7_2d.fastq
[M::main] Real time: 17.636 sec; CPU: 60.304 sec

# layout: empty gfa file
./miniasm -f r7_2d.fastq r7_2d.paf.gz > r7_2d.gfa
[M::main] ===> Step 1: reading read mappings <===
[M::ma_hit_read::1.021*0.99] read 664850 hits; stored 219221 hits and 16136 sequences (50720456 bp)
[M::main] ===> Step 2: 1-pass (crude) read selection <===
[M::ma_hit_sub::1.049*0.99] 11922 query sequences remain after sub
[M::ma_hit_cut::1.055*0.99] 206693 hits remain after cut
[M::ma_hit_flt::1.059*0.99] 206477 hits remain after filtering; crude coverage after filtering: 10.55
[M::main] ===> Step 3: 2-pass (fine) read selection <===
[M::ma_hit_sub::1.069*0.99] 11026 query sequences remain after sub
[M::ma_hit_cut::1.074*0.99] 202966 hits remain after cut
[M::ma_hit_chimeric::1.078*0.99] identified 1 chimeric reads
[M::ma_hit_contained::1.083*0.99] 756 sequences and 556 hits remain after containment removal
[M::main] ===> Step 4: graph cleaning <===
[M::ma_sg_gen] read 541 arcs
[M::main] ===> Step 4.1: transitive reduction <===
[M::asg_arc_del_trans] transitively reduced 97 arcs
[M::asg_arc_del_multi] removed 0 multi-arcs
[M::asg_arc_del_asymm] removed 2 asymmetric arcs
[M::main] ===> Step 4.2: initial tip cutting and bubble popping <===
[M::asg_cut_tip] cut 547 tips
[M::asg_pop_bubble] popped 0 bubbles and trimmed 0 tips
[M::main] ===> Step 4.3: cutting short overlaps (3 rounds in total) <===
[M::asg_arc_del_short] removed 0 short overlaps
[M::asg_arc_del_short] removed 0 short overlaps
[M::asg_arc_del_short] removed 0 short overlaps
[M::main] ===> Step 4.4: removing short internal sequences and bi-loops <===
[M::asg_cut_internal] cut 0 internal sequences
[M::asg_cut_biloop] cut 0 small bi-loops
[M::asg_cut_tip] cut 0 tips
[M::asg_pop_bubble] popped 0 bubbles and trimmed 0 tips
[M::main] ===> Step 4.5: aggressively cutting short overlaps <===
[M::asg_arc_del_short] removed 0 short overlaps
[M::main] ===> Step 5: generating unitigs <===
[M::main] Version: 0.2-r137-dirty
[M::main] CMD: /home/aechchik/wgets/miniasm/miniasm -f r7_2d.fastq r7_2d.paf.gz
[M::main] Real time: 1.920 sec; CPU: 1.745 sec

Just to let you know, I tried the very same commands with the toy example on [PB data](wget -O- http://www.cbcb.umd.edu/software/PBcR/data/selfSampleData.tar.gz) (in your readme) and the gfa file is not empty.

Do you happen to know what is the source of this unexpected gfa file?

Thanks in advance for your help, Amina

[lh3]

Miniasm assumes you are assembling genomic data. It is probably discarding RNA contigs because they are usually too short and/or composed of too few reads. You are entering an unexplored area.

Is it possible to share the reads or just the PAF file with me, so that I can tune the parameters a bit at least to get something (right or wrong) out?

[aechchiki]

Hi @lh3, thanks for your superquick reply! Sure I can share the reads/paf with you. (edit: I found your e-mail) I am sending them to lh3@me.com

Many thanks, Amina

[lh3]

@aechchiki Thanks a lot for the test data. You can run minimap as usual, and invoke miniasm like this:

miniasm -2S6 -f r7_2d.fastq.gz mapping.paf > output.gfa

The output is not empty, but I have little idea whether that makes sense. I would be curious to know if such assembly is usable at all.

[wjyzidane]

I have the same issue and used "-2S6" option but does not work. Are there any other options I can try? Thanks!

[abdullah314]

@aechchiki Thanks a lot for the test data. You can run minimap as usual, and invoke miniasm like this:

miniasm -2S6 -f r7_2d.fastq.gz mapping.paf > output.gfa

The output is not empty, but I have little idea whether that makes sense. I would be curious to know if such assembly is usable at all.

I can't find the option -2S6 in usage section of the software. Please explain '-2S6' .

[HAHasani]

yes pls, could you explain what this flag represents? in my case it did work and produced output

[EmilioKolo]

I had the same problem and it was apparently solved by the "-2S6" flag (I used it on a known sequence and it worked just fine for that). Still couldn't find any explanation of what it does in the documentation nor forums.