Today

1. Today • Blue/white screening • PCR.ppt • Illumina genome assembly • P3S, (A?) • Chromatogram editing

2. Today • HK • B/W screening • *.ppt • Illumina sequencing • P3S Assembly MITOBIM • A, parasite? Assembly MITOBIM • Sequence editing and BLAST data quality, improve, identity of snails Select clones

3. SNAILAND PARASITES BIOLOGY DNA “identity, possibilities” phylogenetics CTAB/DNAzol CTAB/DNAzol Illumina (full) genome sequencing gel electrophoresis nanodrop spec PCRrDNA/mito Qubit Fluorometry Covaris fragmentation Ampure (fragment collection) Kapa DNA library preparation kit Pippin size selection QC Bioanalyzer, Qubit, qPCR Illumina run TA cloning, B/W screening electrophoresis Qiagen plasmid extraction Restriction digests direct sequencing M13 sequencing Sequence ID (BLAST) editing Galaxy QC Data file (MT) genome assembly Mitos, manual annotation Gene annotation Primer design, walking Phylogenetics GenBank submission

4. Pick 2 White colonies/group mark plate of origin on the board and in your notes

5. Friday Each group Select 2 white colonies, sample by “scraping” with yellow tip, Colony that you sampled with a sharpie to avoid resampling Eject yellow tip with bacteria in 2.5 ml LB, 100mg/ml kanamycin MARK TUBE with group number, plate designation (17, 28, 39) Colony number for your group (1 or 2) Either e.g. 9_39_2SHARE PLATES AS NEEDED

6. PCR.ppt

8. NextSeq Illumina run, 130 million, 2 x 150 (300nt) Paired End Reads Result: ≤ 3,900,000,000 nucleotides(inspect all nts @1/sec:/365/24/60/60 = ~123 years) Sequence quality: Trimming Filtering to remove adaptors, barcodes Assembly https://www.youtube.com/watch?annotation_id=annotation_1533942809&feature=iv&src_vid=HMyCqWhwB8E&v=fCd6B5HRaZ8

9. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3711436/pdf/gkt371.pdfhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3711436/pdf/gkt371.pdf

11. MITOBIM

12. P3S SP3 but A? Parasite?

13. EXECUTE MITOBIM FIRST INTERLEAVE, THEN EXECUTE MITOBIM

14. DNA SANGER SEQUENCING “*.ABI” Sequence analysis?

15. Forward primer sequencing reaction sequencing generates + strand ATCG ggaa 5’ 3’ dye blobs Reverse primer sequencing reaction CCTT tagc 5’ generates - strand 3’ dye blobs

16. Chromatograms • EDIT: look to see whether you can do better than the computer algorithm. • Evaluate the peak pattern to Insert, delete, reassign residues • Retrieve sequence from chromatograms • BLAST F and R sequences toward identification of species of origin

17. Possible outcomes NNNNN NNNNNNNNNNNNNNNNNNNNNNNANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGGNNGNNNNNNNNNNNNNNNANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGNNNNNNGGGGGGGNGNGGNNNGGNNNNNNNNNNNNNNNNNA Failed reactions: due to Dead chemistry (BigDye, primers) Contaminants (inhibitors) Salt/Organics Too much/little template (wedge) Loss of extension products (precipitation, running)

18. NNNNNNNNNNNNNNNNNNNNNNNNNNNGNTNNNGNAAGTNNNNNNNNNNNNNGGTACNANNNNNNNNNNNNNNNNNTNTTNTCCNNANGGTAATTTANNNNNNNNNNNNCNNCNNATANNNNNCATAAAATTTTTTTAATAAAATTAGAAAAGTTTCTTTTAAGTTTTTGNNNNNNNGNNNNNCCAACAAAAAATTAGGATGTAATCTATTTTTTCTATTTAAAAAAATGTTATACACTTTTCTTTAAAAATTCTAAGGGTCTTCTCGTCTTTTTTCTAAATTACTGGTATTTTCACCAGATAAACAAATTAAAAAAACACTAATTATTATAGCTACTATTCATTACTTCTTTCATTCCAGACTACAATTAATAGCCAATTGATTATGCTACCTTAGCACAGTCAAGGTACTGCGGCCGTTAATAAAGTTACACCGGGCAGAAGATATCAATAATCTTTTAAAAAATTTTCTACTGACTATGTTTNNNNNAAACAGGCGANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGNTNNNGNAAGTNNNNNNNNNNNNNGGTACNANNNNNNNNNNNNNNNNNTNTTNTCCNNANGGTAATTTANNNNNNNNNNNNCNNCNNATANNNNNCATAAAATTTTTTTAATAAAATTAGAAAAGTTTCTTTTAAGTTTTTGNNNNNNNGNNNNNCCAACAAAAAATTAGGATGTAATCTATTTTTTCTATTTAAAAAAATGTTATACACTTTTCTTTAAAAATTCTAAGGGTCTTCTCGTCTTTTTTCTAAATTACTGGTATTTTCACCAGATAAACAAATTAAAAAAACACTAATTATTATAGCTACTATTCATTACTTCTTTCATTCCAGACTACAATTAATAGCCAATTGATTATGCTACCTTAGCACAGTCAAGGTACTGCGGCCGTTAATAAAGTTACACCGGGCAGAAGATATCAATAATCTTTTAAAAAATTTTCTACTGACTATGTTTNNNNNAAACAGGCGANNN END?? ALSO see: http://seqcore.brcf.med.umich.edu/doc/dnaseq/interpret.html

19. http://www.nucleics.com/DNA_sequencing_support/DNA-sequencing-failed-reaction.htmlhttp://www.nucleics.com/DNA_sequencing_support/DNA-sequencing-failed-reaction.html ALSO see: http://seqcore.brcf.med.umich.edu/doc/dnaseq/trouble/badseq.html

21. Edit, analyze direct sequences, previously edited with Chromas, 16SAr: 5'- CGC CTG TTT ATC AAA AAC AT -3’ 16SBr: 5'-CCG GTC TGA ACT CAG ATC ACG T -3’LCO1490: 5'-GGT CAA CAA ATC ATA AAG ATA TTG G -3’ HC02198: 5'-TAA ACT TCA GGG TGA CCA AAA AAT CA-3’ Download and install codoncode (http://www.codoncode.com/) Activate free trial for 1 team member Import your edited chromatograms into Codon codes AND assemble: (Open codon code); create new project; file; import; add samples (select) Click on unassembled samples; select F and R; contig, assemble; click on contig1 Click in consensus, click button ‘show trace window” (expand if needed) Now scroll through and edit ambiguities as possible. Save, Click in consensus select sequence with and without primers and perform BLASTN your sequences BLASTX your sequence (use translation code 5, invertebrate mitochondrial for snail) Note the genus and species, gene and e-value of your highest hit, with and without the primers. Your previous sequencing included the following reactions: can you assemble the forward and reverse for each of these genes?