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PhilS's Content

There have been 9 items by PhilS (Search limited from 19-September 18)


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#69346 Ouput of MUSCLE alignment shortens names, how can I get the long names back agai

Posted by PhilS on 04 May 2010 - 08:50 PM in Bioinformatics and Biostatistics

GREAT, thanks so much
Phil

Hi,

You unfortunately can't do much about this directly with muscle or many other phylogenetic programs, however you can make it easier to deal with.

Enter REFGEN and TREENAMER, these are two programs I have written in my spare time - read the paper here - they take the standard headers from NCBI GenBank and DOE JGI Genome Project which carry a lot of unhelpful information for your resulting trees (and which cannot be handled by most phylogeny programs as you have noticed) and creates an ID from the accession and species name.

This is obviously shortening the header again but once you have a tree from your analysis, you can use the second tool to replace the ID code with species name and/or accession which are the important parts of the header...

Hope that helps...




#51809 Ouput of MUSCLE alignment shortens names, how can I get the long names back agai

Posted by PhilS on 17 December 2009 - 11:50 PM in Bioinformatics and Biostatistics

Hello,
I'm aligning a bunch of sequences with MUSCLE by inputting a FASTA file with regular header info such as this:
>gi|280987219|gb|GQ200200.2| Cohaesibacter sp. DQHS-21 16S ribosomal RNA gene, partial sequence

However, the ouput file from MUSCLE shortens the names, to something like this: gi|2809872. This seems to be a common thing in phylogenetics software too (I think the phylip format has a short name as well). I presume this is so that reference ID's are passed around the program instead of the full name. But what is the easiest way to get the names back again? For example, I'm feeding the MUSCLE alignment file into RaxML, which creates a tree file (I'm not sure what format this is in) and then I want to look at the names on the branches, not just an ID.

Any help much appreciated,
Thanks,
Phil



#49128 Making trees from 16s of length 1200 bp and 1400 bp, do I need to trim all to 12

Posted by PhilS on 02 December 2009 - 05:47 PM in Bioinformatics and Biostatistics

Thanks,
good website,
Phil

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.




#49095 Making trees from 16s of length 1200 bp and 1400 bp, do I need to trim all to 12

Posted by PhilS on 02 December 2009 - 01:01 PM in Bioinformatics and Biostatistics

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



#49072 Making trees from 16s of length 1200 bp and 1400 bp, do I need to trim all to 12

Posted by PhilS on 02 December 2009 - 11:12 AM in Bioinformatics and Biostatistics

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



#48622 PCR set-up calculation nightmares.

Posted by PhilS on 30 November 2009 - 09:06 PM in PCR, RT-PCR and Real-Time PCR

Many thanks,
I found a bug in my spreadsheet that was causing the errors, I'd always end up with less and couldn't figure it out before.
cheers for the tips and help,
Phil



#48104 PCR set-up calculation nightmares.

Posted by PhilS on 27 November 2009 - 03:48 PM in PCR, RT-PCR and Real-Time PCR

Hi,
I never look forward to setting up PCR reactions, so I'm trying to make it as easy as possible. The problem comes when I have multiple things to set up, for example, the worst situation would be 4 templates, 3 primer sets, 5 temperatures. I've set these up before, but they take a fair bit of planning to get the right calculations for each mastermix (e.g a mastermix for each template, each primer set, then put these into 5 tubes for each of the 5 temperatures). I normally screw up somewhere, particularly with the excess I calculate for each mastermix, wither a lot over or under. I normally use a spreadsheet but I have to change it each time depending on the number of variables, so I was thinking of setting up a number of spreadsheets e.g.

Spreadsheet configuration: mastermixes
x templates, x
x templates, z primers, x + z
x templates, y temperatures, z primers x + y + z

I generally have math phobia although the math is not hard, but when I started to set this up I wondered if I'm over thinking the problem, and wanted to see what others do. Maybe I should forget doing the optimal multiple mastermixes and just pipette the templates and primers into the final tubes themselves, and then prepare one mastemix and add to each. What do you do, or what would you do in this situation?

cheers,
Phil



#45627 Autoclaved water with white flecks in it?

Posted by PhilS on 17 November 2009 - 10:42 AM in General Lab Techniques

Thanks all for notes, I think autoclaving in glass and pipetting into tubes in hood is my best bet.

white flakes in water can be salts which appear to be on the surface after boiling, Can u just boil the same water in a beaker and allow it to cool and check if it appears over the surface...If so, I would get my filter checked first!!


@gogreen, so you are saying it could be salts forming, (I think this is most likely) and if aI boil it then it should go away, otherwise it is something to worry about? Can you explain why would salts appear on the surface after boiling though?


Phil



#45358 Autoclaved water with white flecks in it?

Posted by PhilS on 16 November 2009 - 05:22 PM in General Lab Techniques

Hi,

I prepare my own sterile water for PCR reactions. Every few months. I autoclave ~50 x 1.5 mL eppendorf tubes, each with 1 mL of water inside. The tubes are clean and the water has come from a filtered water source in our lab. When I autoclave, I cover the tops of the tubes to prevent them bursting open. The eppendorf's are then stored in a screw top jar on my bench.

However, every time, after a month or so, I start to see small flecks of white appear in the water. Before I ignored it, knowing that they were still sterile, but as I've been having trouble with contamination recently, I started to suspect this might be the source. But when I look at these specs under the scope, I do not see any cells. I'm thinking of calling the manufacturer of the tubes to ask them for their advice, but I thought I'd post here first.

Any thoughts?

Cheers,
Phil




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