[hakattack]

Thank you Maria. This is a big help for me. Taking your advice, here is what I did

veleth auto 25 -fastq -shortPaired1 myRef_shuffled.fq
velvetg auto_25 -exp_cov auto -ins_length1 150
I get
Final graph has 2 nodes and n50 of 1, max 1, total 2, using 0/10148414 reads

However, I get an empty contig.fa.

Now when when I try kmer 21,
velveth auto 21,25,3 -fastq -shortPaired1 myRef_shuffled.fq
velvetg auto_21 -exp_cov auto -ins_length1 150

I get some contigs, however, they do not align with my reference sequence (500 bases).

Do you see anything wrong I might be doing? I should get some contigs that align with the reference 100%.

[mastal]

How to set -max_coverage in velvetg

the assembly with k=25 won't give you any contigs because velvet has a default min contig size, and contigs smaller than that won't be reported in the contigs.fa file.

you should probably try a wider range of kmer sizes initially, just run velvetg without any parameters other than the directory with the velveth results, then do more runs of velvetg using whichever kmer length or lengths look more promising. Hopefully if you manage to get a better assembly you will get something that aligns to your reference.

[hakattack]

Thank you Mastal,

I have done what you suggested and I get a better luck than before. In one case, I got 100% when I tried k=19 but in another case, I tried all the ks= 15,17,19,21,23 and the best contig I got was 75% but not more than that so I gave up. I am assuming I should not expect better outcomes with such short reads, would you agree?

Thanks