Nov 20, 2020

Public workspaceGeneral Assembly and Alignment in Geneious

DOI
dx.doi.org/10.17504/protocols.io.bnvfme3n
  • 1University of Florida
  • Protocols Bark Beetle Mycobiome
Bark Beetle Mycobiome Research Coordination Network Bark
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DOI: dx.doi.org/10.17504/protocols.io.bnvfme3n
Document Citation: Andrew J. Johnson, Demian F Gomez 2020. General Assembly and Alignment in Geneious. protocols.io https://dx.doi.org/10.17504/protocols.io.bnvfme3n
License: This is an open access document distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Created: October 23, 2020
Last Modified: November 20, 2020
Document Integer ID: 43655
Abstract
The purpose of this protocol is to conduct general assembly and alignment of sequences in Geneious.

This protocol is part of the Bark Beetle Mycobiome (BBM) Research Coordination Network. For more information on the BBM international network: Hulcr J, Barnes I, De Beer ZW, Duong TA, Gazis R, Johnson AJ, Jusino MA, Kasson MT, Li Y, Lynch S, Mayers C, Musvuugwa T, Roets F, Seltmann KC, Six D, Vanderpool D, & Villari C. 2020. Bark beetle mycobiome: collaboratively defined research priorities on a widespread insect-fungus symbiosis. Symbiosis 81: 101–113 https://doi.org/10.1007/s13199-020-00686-9.
  1. Import .ab1 files to Geneious
  2. Assemble - join sequences fwd and rev in one /de novo assembly
  3. Check forward and reverse are ok (can be done during the alignment): rev is X primer, if not, RC button / save
  4. Extract from Assemble consensus: right click on sequences / Generate Consensus Sequences 
  5. Export sequences as fasta file
  6. Trim sequence - Trim with 0,01 limit of probability error. Also, add to trim out PCR primers on ends if sequenced.
  7. Rename / batch rename
  8. Group sequences into a list (right click) to export as fasta file
  9. MAFFT alignment - automatic directions.
  10. Check Frame to avoid Stopping Codons. Set: Invertebrate mitochondrial genome
  11. Create Fast tree or NJ

To concatenate sequences: tools/concatenate, create separate alignments before and name then with the record number