[aharkess]

SSPACE combining cDNA and PE/MP

Hi all,

I'm using SSPACE with a wealth of data, from small PE libraries up to 20kb and 40kb mate pair libraries. In addition, I have three lanes of 2x100nt RNAseq which I'm curious if could be incorporated. My genome is highly repetitive (70%) , so I'm hoping that the more gene space sequence, the better.

I've seen the nematode paper where RNApath was used to scaffold a genome with RNAseq reads, but has anyone successfully used cDNA + PE/MP WGS data in SSPACE? There are some obvious considerations with splicing, but perhaps the plus/minus insert size error can take this into account?

Thanks,
Alex

[sheepyuan]

No, this won't work, since both reads of a pair should be mapped along the contigs. You better make paired-end data by splitting the reads. For example if your read is 200 long, you can make a paired-end read of the first 100bp and the last 100bp. Specify your insert size as 200bp. I've never done this, but I think this could work.

Regards,
Boetsie

Thank you very much, I'll try your method of splitting the read!

[boetsie]

No, this won't work, since both reads of a pair should be mapped along the contigs. You better make paired-end data by splitting the reads. For example if your read is 200 long, you can make a paired-end read of the first 100bp and the last 100bp. Specify your insert size as 200bp. I've never done this, but I think this could work.

Regards,
Boetsie

hi,
I have a question.I have some single-end 454 data, how would the SSPACE run if I artificially make it a pair-end data whose sequence of the other side is all "NNNNNNNNNN"?

[sheepyuan]

hi,
I have a question.I have some single-end 454 data, how would the SSPACE run if I artificially make it a pair-end data whose sequence of the other side is all "NNNNNNNNNN"?

[boetsie]

Hi,

I can't really judge that, since it depends on what you think is 'better'. Anyway, if you have a nice draft assembly, I would not use the contig extension option, main reason is that it is a time and memory-consuming process. Our current strategy is to to use SSPACE for generating the scaffolds followed by our tool GapFiller to close the gaps (N's) produced by SSPACE. GapFiller uses local information from the paired-read data for the extension, instead of all the unaligned reads. This extension is much faster and more reliable.

Regards,
Boetsie

Hi,

Using SSPACE, will it always be better to do contig extension prior to scaffolding? And do I do extension with both paired end and single end reads, or just paired end?

Thanks.

[boetsie]

I'm not really sure what you mean. You could just add your region of alignment in the tab-file, e.g. if the BAC aligns from contig 1 at position 1000-3000 and at contig 2 at position 4000-2000 (so reverse), you can just add this info:

contig1 1000 3000 contig2 4000 2000

SSPACE can only handle links between two contigs, so if a BAC aligns on multiple contigs you have to split it so you only have only a contig-contig link, instead of contig-contig-contig.

Regards,
Boetsie

for the TAB delimited format like:

<contig1> <startpos_on_contig1> <endpos_on_contig1> <contig2> <startpos_on_contig2> <endpos_on_contig2>

E.g.
contig1 100 150 contig1 350 300
contig1 4000 4050 contig2 110 60

if startpos greater than endpos means the reads mapped on to the - strand


I map my BAC end reads on the congits by BLAT , how do I contain the strand information in my TAB files?

[mht]

Hi,

Using SSPACE, will it always be better to do contig extension prior to scaffolding? And do I do extension with both paired end and single end reads, or just paired end?

Thanks.

[biocomfun]

for the TAB delimited format like:

<contig1> <startpos_on_contig1> <endpos_on_contig1> <contig2> <startpos_on_contig2> <endpos_on_contig2>

E.g.
contig1 100 150 contig1 350 300
contig1 4000 4050 contig2 110 60

if startpos greater than endpos means the reads mapped on to the - strand


I map my BAC end reads on the congits by BLAT , how do I contain the strand information in my TAB files?

[boetsie]

It will generate a ‘n’ if a negative gap was found, meaning that there is potential overlap between the contigs but SSPACE could not find a full overlap.

It will generate a lower-case ‘acgt’ if there is actually an overlap found, e.g.;

Ctg1: AGTAGATAGATGATCGCGCTGA
Ctg2:.............ATCGCGCTGAAGTAGATAGATGAGATCGAC

Will be;
AGTAGATAGATGatcgcgctgaAGTAGATAGATGAGATCGAC

Regards,
Boetsie

[mht]

oops boetsie, my bad. they were lower-case ACGTN characters. I used Velvet as my assembler so the lower case acgts were from there. What is the difference between 'n' and 'N' characters in SSPACE?

Hi mht,

SSPACE only adds 'n' or 'N' characters to the assembly, so it would be strange if there are other characters included after scaffolding. Could you please show me an example of what non-ACGTN characters there are included?

Regards,
Boetsie

[boetsie]

Hi mht,

SSPACE only adds 'n' or 'N' characters to the assembly, so it would be strange if there are other characters included after scaffolding. Could you please show me an example of what non-ACGTN characters there are included?

Regards,
Boetsie

Hi Boetsie

I'm trying to scaffold a set of contigs from a bacterial genome assembly. Before scaffolding, there were no non-ACGTN bases in my assembly, but after scaffolding with SSPACE, there were. Can you please let me know what is causing this, and if there is an option to turn it off?

I have already set the -x parameter to 0 to turn off extension.

Thanks.

[mht]

Hi Boetsie

I'm trying to scaffold a set of contigs from a bacterial genome assembly. Before scaffolding, there were no non-ACGTN bases in my assembly, but after scaffolding with SSPACE, there were. Can you please let me know what is causing this, and if there is an option to turn it off?

I have already set the -x parameter to 0 to turn off extension.

Thanks.

[rpdias]

Thanks Boetsie!

Cheers,
Ricardo

Hi Ricardo,

look at this post where colindaven suggests how to fix the problem;

http://seqanswers.com/forums/showpost.php?p=50912&postcount=114

Simply chmod a+x all directories of SSPACE.

Regards,
Boetsie

[boetsie]

Hi Ricardo,

look at this post where colindaven suggests how to fix the problem;

http://seqanswers.com/forums/showpost.php?p=50912&postcount=114

Simply chmod a+x all directories of SSPACE.

Regards,
Boetsie

[rpdias]

Greetings,
I'm quite new to SSPACE. I'm using it in a Ubuntu 64bits VM machine with perl and python.
I'm tying to use the example data to test SSPACE, but I have always the same error, it seems that can't do bowtie indexing. The same happens with original Illumina data.

Anyone had the same problem or can help me on this?

Thanks in advance for any help!

Cheers,
Ricardo


SSPACE Log
perl /media/NGStorage/NGS/SW/SSPACE-BASIC-2.0_linux-x86_64/SSPACE_Basic_v2.0.pl -l libraries.txt -s contigs_abyss.fasta -k 5 -a 0.7 -x 0 -b ecoli_scaffolds_no_extension

Legacy library getopts.pl will be removed from the Perl core distribution in the next major release. Please install the separate libperl4-corelibs-perl package. It is being used at /media/NGStorage/NGS/SW/SSPACE-BASIC-2.0_linux-x86_64/SSPACE_Basic_v2.0.pl, line 87.
Your inserted inputs on [SSPACE_Basic_v2.0_linux] at Tue Aug 21 10:09:36 2012:
Required inputs:
-l = libraries.txt
-s = contigs_abyss.fasta
-b = ecoli_scaffolds_no_extension

Optional inputs:
-x = 0
-z = 0
-k = 5
-a = 0.7
-n = 15
-T = 1
-p = 0


=>Tue Aug 21 10:09:36 2012: Reading, filtering and converting input sequences of library file initiated
Reading read-pairs lib1.1 @ 10000000

------------------------------------------------------------

=>Tue Aug 21 10:10:11 2012: Storing contigs to format for scaffolding

LIBRARY lib1
------------------------------------------------------------

=>Tue Aug 21 10:10:12 2012: Reading contig file

=>Tue Aug 21 10:10:12 2012: Building Bowtie index for contigs

Bowtie-build error; -1 at /media/NGStorage/NGS/SW/SSPACE-BASIC-2.0_linux-x86_64/bin/PairingAndScaffolding.pl line 829.
**************************************************

Process failed on Tue Aug 21 10:10:12 2012