Geneious for transcriptome alignment

toreoe · 01-31-2013, 05:13 AM

Hi there,

I am new to NGS data and bioinformatics. But SEQanswers seems like a good place to start. Here is my problem for you :-)

Computer resources: Geneious R6, access to cluster with various bioinf programs
Data: 454 GS FLX transcriptome reads from 12 individuals, two species
Goal: Find overlapping sequences from orthologous genes within and between species

I have 454 transcriptome reads from two species (6 individuals from each -->12). We want to do a comparative transcriptome project, studying intra- and interspecific variation and divergence patterns, selection signs, etc. So, what we want is to strip the dataset down so that it contains overlapping sequences from orthologous genes, preferentially utilizing all individuals.

I am no wizard in bioinformatics. I am at a level where peeking at the post-it notes filling my wall containing basic UNIX commands (ls -l, chmod, less, etc.) is still very much useful. But with good help I've been able so far to assemble the 454 reads for all 12 individuals using Newbler 2.6. What I hope now is that our newly purchased Geneious Pro (R6) can help in order to 1) align the individual transcriptome contigs to each other 2) strip down dataset to overlapping sequences, if there exists any (apparently the coverage is variable).

Preferentially, I hope the Geneious users out there can help instruct me on how to go forward with this. But any suggestion will be much appreciated, also advise for alternatives to Geneious.

One thing I have tried is to concatenate the largest individual assembly and using the map to reference option in Geneious to align another individual assembly to it. Any comments on this approach?

Best wishes,
Tore

Geneious · 01-31-2013, 08:26 PM

Hi Tore,

You could try annotating your reference sequence with coverage annotations for each sample then compare the annotations to find overlapping regions.

You could also try de novo assembling your contigs from every sample together, probably with the "Don't merge contigs when there is a variant with coverage over ..." setting turned off. Doing that will also mix paralogs in with the orthologs you're after but it might be helpful.

Also try checking out the support pages for Geneious for more advice on using Geneious.
https://support.geneious.com/home

Kind regards,
Helen Shearman
Geneious Support Analyst
www.geneious.com

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01-31-2013, 05:13 AM	#1
toreoe Junior Member Location: Norway Join Date: Jan 2013 Posts: 3	Geneious for transcriptome alignment Hi there, I am new to NGS data and bioinformatics. But SEQanswers seems like a good place to start. Here is my problem for you :-) Computer resources: Geneious R6, access to cluster with various bioinf programs Data: 454 GS FLX transcriptome reads from 12 individuals, two species Goal: Find overlapping sequences from orthologous genes within and between species I have 454 transcriptome reads from two species (6 individuals from each -->12). We want to do a comparative transcriptome project, studying intra- and interspecific variation and divergence patterns, selection signs, etc. So, what we want is to strip the dataset down so that it contains overlapping sequences from orthologous genes, preferentially utilizing all individuals. I am no wizard in bioinformatics. I am at a level where peeking at the post-it notes filling my wall containing basic UNIX commands (ls -l, chmod, less, etc.) is still very much useful. But with good help I've been able so far to assemble the 454 reads for all 12 individuals using Newbler 2.6. What I hope now is that our newly purchased Geneious Pro (R6) can help in order to 1) align the individual transcriptome contigs to each other 2) strip down dataset to overlapping sequences, if there exists any (apparently the coverage is variable). Preferentially, I hope the Geneious users out there can help instruct me on how to go forward with this. But any suggestion will be much appreciated, also advise for alternatives to Geneious. One thing I have tried is to concatenate the largest individual assembly and using the map to reference option in Geneious to align another individual assembly to it. Any comments on this approach? Best wishes, Tore Last edited by toreoe; 01-31-2013 at 05:39 AM.

01-31-2013, 08:26 PM	#2
Geneious Registered Vendor Location: New Zealand Join Date: Jul 2010 Posts: 22	Hi Tore, You could try annotating your reference sequence with coverage annotations for each sample then compare the annotations to find overlapping regions. You could also try de novo assembling your contigs from every sample together, probably with the "Don't merge contigs when there is a variant with coverage over ..." setting turned off. Doing that will also mix paralogs in with the orthologs you're after but it might be helpful. Also try checking out the support pages for Geneious for more advice on using Geneious. https://support.geneious.com/home Kind regards, Helen Shearman Geneious Support Analyst www.geneious.com Last edited by Geneious; 01-31-2013 at 08:28 PM. Reason: Typo