Scaffolding despite multiple links in SSPACE

[swe]

I have denovo assembled a 1.8 Mbp genome with a PE 100 bp 360 insert library using abyss resulting in 69 contigs. After that I scaffolded these contigs with a MP 100 bp 3300 insert library using SSPACE-Premium resulting in 7 scaffolds.

My problem is that SSPACE makes connections between contigs although the contig link to multiple other contigs. This is the command I used for running SSPACE.

SSPACE_Premium_v2.2.pl -l libSSPACE.txt -s contigs.fna -k 20 -a 0.9 -x0 -b outdir -T 12 -p1

libraryfile libSSPACE:
lib4000 bwa rawseq_MP_1.txt.gz rawseq_MP_2.txt.gz 3356 0.2 RF

When running this I get a connection between contig 37 and contig 15 although contig 37 has links to 4 different contigs.

r37 has 1118 links with r14 and gap of 351 bases
r37 has 1051 links with r36 and gap of 488 bases
r37 has 595 links with f12 and gap of 334 bases
f37 has 542 links with f15 and gap of -242 bases

I guess that this depends on the building scaffolds part with the two different two methods/ratios for generating reliable scaffolds.

I do not trust these results and how can I set the parameters to get reliable scaffolds with my long insert library?

[seb.lees]

Hi swe,

SSPACE choose the 'best' contig connection according to the number of links between contigs (defined as the number of pairs connecting the two contigs). If your r37 have more links to r15 than to the other contigs, r37-r15 will be chosen as the preferred connection.

The -a parameter controls the threshold difference. To my understanding, the lower the more stringent. For instance, a 0.9 value means that if 10 links connect r37 and r15, and 9 links connect r37 and other contigs, r37 and r15 will be scaffolded because you have a ratio of 0.9. So you could reduce your -a parameter to avoid connections you don't trust into.

See the Suplementary Figure S2 from the SSPACE paper: http://bioinformatics.oxfordjournals...4/578.abstract

However in your case, I don't know whether the ratio will be computed as: links r37-r15 vs links r37-all other contigs concurrently, or as: links r37-r15 vs links r37-each other contig independently.

Seb.