Molecular markers - computer practicals
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Analysis of DNA sequences

Presentation is here.

Training dataset is here. It consists of ten samples from the genus Pericallis sequenced for the trnL-trnF chloroplast region.
The sequences we generated during a lab part of the course will be sent to you by e-mail.

A ZIP file with necessary software is here. Download it, unzip and install some of the software:
FinchTV - ABI format sequence viewer, installation necessary
ClustalX - sequence alignment software, installation necessary
BioEdit - simple alignment viewer/editor with many other functions, installation necessary
RC - reverse-complement maker, just run the exe file
SeqState - insertion/deletion coder, just run the jar file (requires Java)
Gblocks - alignment trimming, just run the exe file
trimAl - alignment trimming, command line use
TCS - statistical parsimony network, just run the jar file (requires Java)

The software Geneious should be downloaded here (14 days free trial version, registration necessary). For later use, our faculty has a network licence... ask me how to set up).

Other software recommended to install

• Notepad++
• Total Commander

Tasks (to work with Pericallis sequences)

1. Look at the sequences in AB1 format using FinchTV

• open the file and scroll through the whole sequence - What is the last position you would consider reliable and why?

• look at the chromatogram info ('i' icon) - What is the instrument name? When it was analyzed?

• look at the raw data (View - Raw Data) - What is the maximum number at vertical axis (these are rfu - relative fluorescence units)? Are there any changes towards the end of sequence?

• BLAST the sequence against on-line NCBI database (Edit - BLAST sequence) - What is the best hit? What is the similarity to it?

2. Prepare contigs using either Geneious or SeqMan

• align the sequences, trim the unreliable ends - How long is the overlap in the middle?

• do the necessary edits (delete gaps, put Ns when the base call is not reliable) and copy the resulting contig to text editor, e.g. Notepad++

• submit the sequence using Google Form (we will allocate the work among us, everybody submits 1-2 sequences, all collected sequences appears here)

3. Align sequences using standalone ClustalX and on-line MAFFT approach

• align sequences in FASTA format using ClustalX - What files are created? Can you create also some other alignment formats?

• align sequences using MAFFT - Did you get the same alignment?

• save alignments as FASTA (gapped FASTA, i.e., aligned sequences)

4. Trim the alignment using Gblocks / trimAl

• open Gblocks and trim the alignment using default settings - How many positions were eliminated and why?

• copy your alignment to trimAl/bin folder, open commandline there (e.g., using Total Commander) and run following commands:

  • .\trimal -in alignment.fas -out out_nogaps.fas -htmlout out_nogaps.html -nogaps #removes all column with at least one gap

  • .\trimal -in alignment.fas -out out_gappyout.fas -htmlout out_gappyout.html -gappyout #removes gappy regions

  • .\trimal -in alignment.fas -out out_strict.fas -htmlout out_strict.html -strict #more strict filtering

• check output files (in html file you can see what was removed) - How many positions are retained in these three trimming cases?

5. Convert FASTA files to PHYLIP and NEXUS format

• use on-line service, e.g. EMBOSS Seqret or Format Converter

• use the commandline option in trimAl/bin folder

  • .\readal -in out_gappyout.fas -out out_gappyout.phy -phylip3.2

  • .\readal -in out_gappyout.fas -out out_gappyout.nex -nexus

• What are advantages of these formats?

6. Simple indel coding (SIC) using SeqState

• run SeqState, open the alignment and select IndelCoder and desired type of indel coding

• open the resulting file - What format it is? How many indels were coded and how?

7. Working with FaBox (on-line fasta  sequence toolbox)

• trim the alignment to the shortest sequence using 'Alignment trimmer' - What it does? What is the length of the resulting alignment?

• extract variable sites only using 'Show variable sites only' - How many variable sites are there?

• generate input file for TCS using 'Create TCS input file from fasta (fasta2tcs)' - Open the resulting file in, e.g., Notepad++ and check 'end-of-line' characters. Are there different from the input file? How?

8. Generating statistical parsimony network using TCS

• open the JAR file, start new analysis, select PHYLIP file generated in the previous step, change gaps to missing

• look at the resulting graph - What are the small dots between haplotypes? What do you think the network shows?

Good luck and thank you for joining the practical course...