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Plant DNA Barcoding: data workflow

Plant DNA Barcoding: data workflow. Aron Fazekas University of Guelph . Plant DNA Barcoding: data workflow. Workflow Outline: raw sequence editing data alignment re-edit the sequence file upload to BOLD quality checks using BOLD / genbank. Sequence editing: primer trimming.

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Plant DNA Barcoding: data workflow

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  1. Plant DNA Barcoding: data workflow Aron Fazekas University of Guelph

  2. Plant DNA Barcoding: data workflow Workflow Outline: raw sequence editing data alignment re-edit the sequence file upload to BOLD quality checks using BOLD / genbank

  3. Sequence editing: primer trimming

  4. Sequence editing: primer trimming 5’ GTTATGCATGAACGTAATGCTC GAGCATTACGT….

  5. Sequence editing: primer trimming

  6. Sequence editing: editing miscalls

  7. Sequence editing: congruence between forward/ reverse reads

  8. Sequence Alignment After editing: need to align the data Kelchner (2000) Ann Missouri Bot Gard rbcL easy to align - most programs work well matK tricky to align – TransAlign seems to do the best job trnH difficult (impossible between genera?) ITS difficult (impossible between genera?) Clustal www.clustal.org TransAlign http://www.biomedcentral.com/1471-2105/6/156 K-Align http://www.ebi.ac.uk/Tools/msa/kalign/

  9. Sequence Alignment Problems to look for after alignment: - primers not trimmed - gaps at the ends - gaps in the middle (protein coding) - translation shows stop codons

  10. - primers not trimmed trnH-psbA Real data submitted for publication - gaps at the ends

  11. rbcL data submitted for publication - gaps in the middle of a coding region

  12. Translate coding regions (rbcL, matK) to ensure there are no stop codons present

  13. Edit both the alignment file and the original sequence file

  14. Can trnH-psbA (or other non-coding sequence) be aligned across diverse species?

  15. Upload to BOLD

  16. After data is edited, aligned: use BOLD to create a tree

  17. Check for misplaced taxa – • remove them from the dataset • Check for singleton species – make a list

  18. BOLD BLAST check

  19. Genbank BLAST check

  20. Genbank BLAST check

  21. Genbank Blast

  22. Acknowledgements Sujeevan Ratnasingham & Bold Team

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