5 replies to this topic

[PhilS]

Hi,
I'm sequencing 16s rDNA sequences from a bunch of mixed bacterial cultures with different primer sets to amplify different strains.  Some of the sequences are 1200 bp, others are full length ~1400 bp.    I could get full 1400 bp sequences from all of the strains, but it is an extra expense and time consuming to clone them rather than just sequence the pcr products directly.  My question is this; when it comes to making phylogenetic trees from these mixed 1200 and 1400 bp sequences, I start off by aligning all of my sequences.  Normally I then trim the sequences down to the shortest sequence and then make a tree from these with e.g RaxML.  But this means that I am bound to the shortest sequence, so in this case, I'd be loosing 200 bp from the 1400 bp sequences and all of my sequences would be 1200 bp.   Is this my only option, or can I use sequences of mixed length in my alignments and trees?

Help much appreciated,
cheers,
Phil

[phage434]

If you sequence the pcr products from the center outward, you can get sequence for the beginning and end of the amplified region, avoiding the cloning step.

[PhilS]

phage434, on Dec 2 2009, 12:06 PM, said:

If you sequence the pcr products from the center outward, you can get sequence for the beginning and end of the amplified region, avoiding the cloning step.

That is true and I'd considered this, but I'm using degenerate primers for a particular set of organisms which are only designed for 1200 bp, I don't have the sequences to design them for 1400 unfortunately.   My other option is to use regular primers for the full length 16s and then clone and colony pick  until I find the sequences of those organisms.  

So my question is, can I use partial sequences in an alignment / tree, or do I have to trim to the shortest sequence that I have?

cheers

Phil

[phage434]

I'm sure you can truncate your longer sequences and align; but if you have time, I would imagine that these universal primers would work for your species:
http://openwetware.o..._identification
If you need a copy of the Lane91 chapter, let me know.

[PhilS]

Thanks,
good website,
Phil

phage434, on Dec 2 2009, 02:13 PM, said:

I'm sure you can truncate your longer sequences and align; but if you have time, I would imagine that these universal primers would work for your species:
http://openwetware.o..._identification
If you need a copy of the Lane91 chapter, let me know.

[HomeBrew]

Have you seen the Ribosomal Database Project at Michigan State University?  Lots of tools and info there...