Step_by_step_class_Workflow.md

Primer design tutorial

1. [Optional] drag zipped tutorial over

2. Start with exercise 5 design of PCR primer pairs

   • They have already downloaded sequences, etc.  But in real life, we would have to get the sequences into Geneious ourselves.

3. Make a separate new folder - NIAID_Training (File -> New Folder)

4. Search NCBI -> Nucleotide for NC_007596

5. Drag sequence to NIAID_Training

6. Familiarize oursevels with the different tabs

   • Sequence view - zoom in to see letters

   • Annotations - tabular view

   • Text view - genbank file

   • Lineage - important later to trace your work

   • Info - can add specified metadata - and make your own fields with Edit

     • click on fields to edit

7. Select COX1 gene - either in the picture or clicking annotation or searching

8. Click Primers

   • Geneious uses Primer3 for primer design - v 2.3.7 modified

   • current version of Primer3 is 2.5.0 released Aug 2019

   • 3 options:

     1. Design New - design based on region

     2. Design with existing - forward primer already have - want to make a reverse primer to go with it

     3. Design for Sequencing – tile amplicons across a region

9. Design New - (Reset to Defaults for the Class - gear bottom left)

   • Task – generic

   • Included region checked - primers will bind inside the region, if you want specific sub-region, put that under target region - primers will not bind in this region

   • Product size – 200-300 (degraded DNA?) - optimal 250

   • Primer - Size Max 24 - Tm Min 54

   • Formula Santa Lucia is newer http://primer3.org/manual.html\#PRIMER\_TM\_FORMULA

   • Characteristics 0 Max Tm Difference 2

   • A mispriming library is a set of sequences (usually repeats) which the primers should not bind to. – Included libs are from Primer3

   • Click OK

10. Click Save

11. Click on Annotations to see primers and characteristics

12. Click on primer set 5498 and 5747 - each primer -> Extract - label COX1

13. Click on the primer sequence in the sequence list at top look at different tabs including Lineage

14. Test primer for specificity of other species - not as good as NCBI Primer BLAST

15. File -> Import -> multiple files -> GeneiousTraining

- DQ316068.gff3 and DQ316068.fasta (Asiatic elephant)

16. Imports as single record

17. Test the primers - select COX1 gene

18. Primers ->Test with Saved Primers

    • Source current folder - Choose - select primers

    • Annotation - check all boxes

    • Region - Find primers that bind inside the selected region

    • Allow 2 mismatches - not same species - no mismatches in last 5bp

19. Zoom in to each primer to see mismatches 

20. Click Save

21. Select both primers, then up top - Primers -> Extract PCR product

    • Extract primer bases

22. Click on PCR product -> Info -> Show OPtions

23. Importing your own set of primers 

    • at least 3 column table - Name Sequence Description

    • tab-delimited or csv

24. File -> Import -> CSV/TSV sequence table - must be saved as .tsv or .csv (not .txt as in Excel)

25. Determine characteristics - will do Tm calculation

Workflows

1. KY616974.1 - import genbank file or search NCBI

2. Click on Workflows – Manage Workflows – New Workflow

3. Build workflow - for each document (will run workflow on each document/sequence/object you have selected) - Test with saved primer

   • extract pcr product

CRISPR Site Finder tutorial

• Good background info https://www.addgene.org/guides/crispr/

• Note that activity (on-target) scoring methods for Cpf1 sites are not currently available.

• Geneious manual chapter: https://assets.geneious.com/manual/2020.0/static/GeneiousManualse79.html

• Cpf1 used for larger regions

• Import yeastgenomes.geneious – will save as yeast_genome folder

• highlight all the sequence/chromosome files - annotations tab - search

  • trp1 and extract the gene

• Click on TRP1 sequence – Cloning - CRISPR Site finder

  • Can change PAM sequence if needed – preview is below