Draft Paper of Beta-Actin Arabian Camel

[MManee]

• it is good to start reading the section of "Materials and methods" with focus on in silico analysis
• please use the sequence (B-Actin Arabian Camel) forwarded to you to identify the following:
  • multiple sequence alignment and analysis of phylogenetic relationships
  • prediction of the secondary and 3D structure of beta-Actin
  • physical and chemical properties of beta-Actin protein
• you will receive an invitation form ShareLatex to be one of the work collaborators to edit the draft paper of Beta-actin Arabian Camel:
  • link to Beta-actin draft: beta-actin draft
  • please don't hesitate to post a question or an enquiry, the team members will be happy to help
• please respect the following Deadlines:
  • [ ] perform & generate results of in silico analysis only: 13 August 2017
  • [ ] write section of results of in silico analysis only: 24 August 2017
  • [ ] write section of methods of in silico analysis only: 1 October 2017
  • [ ] write section of introduction: 12 October 2017

[malsaeed]

I have question regarding the alignment We will cover the mammals same as in the reference paper

Mariam

[SULTAN-ALHARBI]

Hi Mariam, thank you for your question as well as your effort after you translated beta-actin mRNA to protein(Amino acids) sequence BLAST your protein in NCBI protein BLAST ten to twenty most similar to your protein seq will be use to make your phylogenetic tree So it is not good idea to limit your self with the mammals in the reference paper

All The Best Sultan By the way The PCR result of beta-Actin was very good :)

[MManee]

• @malsaeed, please find the first version of submitted paper for HSPB1 arabian camel ijms-217531.pdf
  • it is your main reference paper
  • it will also help you to use the proper tools and generate your figures for beta-actin
• it is recommended that you use geneious software to generate:
  • the multiple sequence alignment of amino acid sequence of arabian camel beta-actin protein
  • secondary structures of arabian camel beta-actin protein
• @SULTAN-ALHARBI please let me know if we still have available license key for geneious software

[SULTAN-ALHARBI]

Sure Here You Are: Download Link: http://www.geneious.com/ Licensee: SULTAN ALHARBI License Key: 1BFA-15C8-99BC-1DF9-60C1 Valid from 07 July 2017 to 07 July 2020

[malsaeed]

@MManee @SULTAN-ALHARBI

Good morning

Hope both doing well Its seems that no available license key I will use the trial version and see after that another solution

Thanks for your effort

[SULTAN-ALHARBI]

Try now, the same licensee

[malsaeed]

same message appear

[SULTAN-ALHARBI]

bring your laptop and let's meet with Dr. Manee at his office!!! When will you be available to meet up?

[malsaeed]

@MManee @SULTAN-ALHARBI My workflow

1-translate the seq from nucleotide to amino acid using (trasnseq-EBI) there is some stars in the translated seq 2- Blast (NCBI) program -PSI balst the seq with the default parameters then I download the result (complete seq) -the result its 8 seq

3- doing the alignment using geneious
4- construct the tree using geneious tree building

5- prediction of secondary structure using - PSIPRED : I have enterd the amino acid seq its show this massage error You must provide a short identifier for your sequence Your sequence contains invalid characters DHIHSTSKGPSLLQLCQPGSTNTSSTWMMVFLCLWLTMALAHARLALRVTMYPMPPSHPSWGTPGTRVSWWAWARRTPMWVMRPRAREASPSSTPWSMASSPTGMTWRRSGTTPSTMSCMWPPKSTPSCPRPPTPRPIVRR*PRSCSRPSTPQPCTWPSMLCCPCTPLAAPLAS*ALVMGPLTLCPLMKGTPSPMQYVTWLAGNRTSSSRSSHSVATASAPQPRGKSCVTSRRSSATSPWTWSRRWPPPPPAPPWRRATSCPTVRSSPSAASASAAPRLSSSPPSWAWKPVASMRLLSTPSSVMWTSSRTSTPTRCCPAGPPCTPATAGCRRSSPPRPPAR*RSRSSRPPRASTPCGWAAPSWPRCPPPSRCGSASKTTTNPAPPSSTASASRRTELPSTVLLTKPNL

does my workflow correct or I did something in a wrong way ?

If we can meet about 2:30 today?

[SULTAN-ALHARBI]

Ok let's meet then

[SULTAN-ALHARBI]

@malsaeed

Ten tissues: Brain, Testis, Liver, Lung, Kidney, Spleen, Stomach, Skin, Heart, Esophagus.

[malsaeed]

@MManee @SULTAN-ALHARBI

Good Morning

The workflow as directed by Dr.manee

1-multiple sequence alignment and analysis of phylogenetic relationships(is done by geneious software)

2-prediction of the secondary and 3D structure of beta-Actin for prediction I follow the help tutorial for geneious and it show me this result for secondary structure

for 3D structure still struggling with geneious there is away but I still try with it

3-physical and chemical properties of beta-Actin protein( almost done I see some tabs in geneious software have some prperties )

hope I do it today

thanks

Mariam alsaeed

[SULTAN-ALHARBI]

@malsaeed @MManee Hi Mariam and Sorry for late reply Let's take it one by one 1- About your multiple Seq alignment result and Phylogenetic trees, it is really good for posting them here so we can further discuss and interpret the results that will help us write in latex :) 2- For the 2nd structure, you predicted 2nd structure of Beta-Actin mRNA if you look back to your result, you will see the icon (RNA fold) which is in your case Beta-Actin mRNA. In fact, we want the 2nd and 3D structures of amino acid sequence of Beta-actin (408 amino acid). A) For 2nd Structure of Beta-Actin protein use (PSIPRED program) this will take about 30 to 90 mins

B) For 3D Structure use 1) Swiss-Model https://swissmodel.expasy.org/interactive or 2) Phyre2 http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index

3) For physical and chemical properties, We will discuss those after we finished 2nd & 3D structures.

All The Best 💯

[SULTAN-ALHARBI]

This is the sequence of Camel_Beta_Actin mRNA

[malsaeed]

@MManee @SULTAN-ALHARBI

Good Evening

The workflow as directed by Dr.manee

1-multiple sequence alignment

analysis of phylogenetic relationships(is done by geneious software)

2-prediction of the secondary and 3D structure of beta-Actin

secondary structure of beta-Actin

cd1cf268-7a7a-11e7-99b5-00163e110593.2d_beta.psipred.pdf

3D structure of beta-Actin

Mariam

[SULTAN-ALHARBI]

@MManee @malsaeed 1) Identify the conserved domains present in a protein sequence of Camel Beta_Actin using https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi

2) Prediction ordered and disordered regions of Beta_Actin protein using GlobPlot server (http://globplot.embl.deis). 3) Calculate isoelectric point (pI) for Arabian Camel Beta_Actin protein using a computer algorithm Beta_Actin Protein (http://isoelectric.ovh.org). 4) physical and chemical properties of beta-Actin protein using those methods [http://tools.iedb.org/bcell/

](url)

# After that we should start writing up

[malsaeed]

Ok got it 👍🏽

[malsaeed]

@MManee @SULTAN-ALHARBI

Good evening

I finished the task and I need to read some topics before I start writing and interpret the result

Mariam

[MManee]

• amazing work, well done @malsaeed
  • please list your results for each in silico analysis
  • don't use screenshots (use format jpg, png, pdf and etc.)
  • this will help us processing your results easily and efficiently

[MManee]

• please do not insert your result figures into Latex file before we process and confirm them here (this GitHub issue)

  please list your results for each in silico analysis don't use screenshots (use format jpg, png, pdf and etc.) this will help us processing your results easily and efficiently

[malsaeed]

@MManee @SULTAN-ALHARBI Sorry for the confusing

Here the results picture

1-Multiple sequence alignment

2-Analysis of phylogenetic relationships(is done by geneious software)

3-Prediction of the secondary and 3D structure of beta-Actin

Secondary structure of beta-Actin

cd1cf268-7a7a-11e7-99b5-00163e110593.2d_beta.psipred.pdf

3D structure of beta-Actin

4-Identify the conserved domains present in a protein sequence of Camel Beta_Actin using https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi

5-Prediction ordered and disordered regions of Beta_Actin protein using GlobPlot server (http://globplot.embl.deis).

6-Calculate isoelectric point (pI) for Arabian Camel Beta_Actin protein using a computer algorithm Beta_Actin Protein (http://isoelectric.ovh.org).

7-Physical and chemical properties of beta-Actin protein using two method [http://tools.iedb.org/bcell/

Mariam

[atalgarni]

Dear Mariam,

Great job you have done so far, Mariam. I want to introduce myself first, This is Abdulmalek Algarni one of KACST_Bioinformatics Group team and it is pleasure having you joining us. The job you have done might need more of attention to the aa sequence used here. I suspect that you did not preprocess the aa for other species before you did the multiple alignment. If so, you might need to reprocess all the species' aa so that all of them start with Methionine amino acid. Please reconsider preprocessing your sequences as highlighted below :

[malsaeed]

Dear @AbuMohammad

Thanks for the great welcoming

I see what you mention and I'm trying to solve it

Thanks

[malsaeed]

Good evening

@AbuMohammad

How can I reprocess all the species' aa so that all of them start with Methionine amino acid? I try and still trying to do it using Geneious software

Thank you

[malsaeed]

what about the above alignment? Is it correct ?

thanks

[atalgarni]

Good evening to you to,,,,

What I meant is that you have to make sure most of your aa sequences start with the right Methionine

For some reasons the sequences you choose might have more than methionine amino acid at the N terminus and are separated by few amino acids so make sure that your aa sequences start with something close to your query sequence in this case the camel beta actin sequence for example Your query sequence start with

MPPP**

And the other species start with

MSTE*MPPA Here the second sequence has more than one methionine at the N terminus ! The correct starting aa is the second Methionine In this example I will delet the aa before MPPA

Just double check all the sequences this would give. Ice multiple alignment of your sequence and would help depicting a nice phylogenetic tree.

All the best

[atalgarni]

This would give nice multiple ***

Sorry texting from my phone

[atalgarni]

In Genious just edit the sequences by deleting some aa

Double click on the sequence of interest Click enable editing Select aa that you want to delete then save changes

[atalgarni]

I think the right Methionine is the aa number 34

MDD***

I might be wrong but this is what I think looking back at your sequence

[SULTAN-ALHARBI]

As my colleague Abdulmalek mentioned, We have to trim the protein multiple alignment sequence before we construct a phylogenetic tree :) Please see the link below for how to do it https://1drv.ms/v/s!ApiRb0qsuCbrgYtD5jMVH3x7cXM_kg If the link does not work immediately, copy and paste it on you favorite internet browser good Luck

[malsaeed]

@SULTAN-ALHARBI @AbuMohammad

((Please ignore this comment I will check every sequence alone )) Good afternoon

I applied the trim for the sequence as directed and got this result

The tree

and Im try to see another soultion

thanks

mariam

[malsaeed]

Hi

I had full search (Automatically + manually)

Searching for the right methionine and I found as Abdulmalek mentioned that is the one begin with

MDD**

I trim and edit the sequences but I got this tree I think its not a good tree

waiting for the responses what I should do?

thanks

[atalgarni]

Hi Fantastic job .... it was tricky sequence you've got here ,, do not worry about the phylogenetic tree ... the topology just represent your multiple alignment which in fact confirms the conservative nature of this protein.

I think you should move on with writing

[malsaeed]

@SULTAN-ALHARBI @AbuMohammad Hi good afternoon

Yes its tricky and so confused I was thinking that its wrong and keep repeating the process

I dont know do you have papers have same tricky tree

and for the rest analysis Should I reprocess with the trimmed camel beta Actin because the trimmed sequence is 375 AA and the translated sequence is 408 AA

thanks

Mariam

[malsaeed]

@MManee
Which picture I can insert it now in latex? Picture for the new Result

Alignment

@AbuMohammad The tree with new representation

Thanks

[MManee]

• good job mariam, based on your preliminary results I think we should investigate different arabian camel gene
• as you identified that the beta-actin is a highly conserved gene across species
• I am going to close this issue and open another one with different gene
• so now you have good experience how to deal with similar in silico analysis, I am expecting to get work done in a short period of time when you work on upcoming gene