1 / 32

DNA Sequencing and Analysis Services for Phylogenetics Studies

We offer a range of DNA sequencing and analysis services, including genome sequencing, PCR, gel electrophoresis, and phylogenetics studies. Our services include primer design, gene annotation, and data analysis using tools like BLAST and Galaxy.

jennadelarosa
Télécharger la présentation

DNA Sequencing and Analysis Services for Phylogenetics Studies

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Today • HK • Primers, sequence inserts • Taxonomic ID 16S direct sequence • *.ppt • Check Mitochondrial Genome Annotation

  2. 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

  3. Not placed 1R, 3 allF4

  4. Primers: Working concentrations: Prepare to 50 mmolar how? Dilute 1/30 how? Labelprimer/30 group number Then add to sequence reactions Components? Group primer Label 1 A0R2 2 A2F2 3 A6R2 4 A7F1 5 BCF1 6 BCR1 7 CCF2 8 CCR2 9 DCF1 10 M13 (already 50 mmolar)

  5. Primers: Working concentrations: Prepare to 50 mmolar how? Label :Nmoles/50 * 100ml Dilute 1/30 how? 2 +58, mix! Labelprimer/30 group number Then add to sequence reactions Components? TRRM 1 5x 2 mastermixed Mq 3 Plasmid 3 Primer 1 Group primer Label 1 A0R2 2 A2F2 3 A6R2 4 A7F1 5 BCF1 6 BCR1 7 CCF2 8 CCR2 9 DCF1 10 M13 (already 50 mmolar)

  6. Label group number, reaction number • Group reaction 1 reaction 2 • plasmid primer plasmid primer • 1 3.2 1 3.2 2 • 2 3.2 3 3.2 4 • 3 3.2 2 3.2 3 • 4 7.1 10 7.1 9 • 5 7.1 1 7.1 2 • 6 7.1 2 7.1 3 • 7 7.1 9 7.1 10 • 8 10.4 5 10.4 8 • 10.4 7 10.4 5 • 10 10.4 6 10.4 7 • Take mastermix tube, label group#, raction# • go to group with plasmid, add 3ml plasmid to each mastermix. • Then go to group with primer number, add 1ml of primer/30. • Spin!, bring to thermal cycler.

  7. Nolan et al, 2014 van Horn et al, 2010

  8. Physidae Lymnaeidae Planorbidae Panpulmonate, non hygrophylid

  9. Physidae Lymnaeidae Planorbidae Panpulmonate, non hygrophylid

  10. Physella acuta Physidae Lymnaeidae Planorbidae Panpulmonate, non hygrophylid

  11. Lineage( full ) cellular organisms; Eukaryota; Opisthokonta; Metazoa; Eumetazoa; Bilateria; Protostomia; Lophotrochozoa; Mollusca; Gastropoda; Heterobranchia; Euthyneura; Panpulmonata; Hygrophila; Lymnaeoidea; Physidae; Physella acuta

  12. Lineage( full ) cellular organisms; Eukaryota; Opisthokonta; Metazoa; Eumetazoa; Bilateria; Protostomia; Lophotrochozoa; Mollusca; Gastropoda; Heterobranchia; Euthyneura; Panpulmonata; Hygrophila; Lymnaeoidea; Physidae; unclassified Physidae

  13. Lineage (full): cellular organisms; Eukaryota; Opisthokonta; Metazoa; Eumetazoa; Bilateria; Protostomia; Lophotrochozoa; Mollusca; Gastropoda; Heterobranchia; Euthyneura; Panpulmonata; Hygrophila; Lymnaeoidea; Lymnaeidae, unclassified Lymnaeidae

  14. Lineage( full ) cellular organisms; Eukaryota; Opisthokonta; Metazoa; Eumetazoa; Bilateria; Protostomia; Lophotrochozoa; Mollusca; Gastropoda; Heterobranchia; Euthyneura; Panpulmonata; Hygrophila; Lymnaeoidea; Ancylidae, unclassified Ancylidae

  15. *.ppt

  16. Nolan et al., 2013 SO: redo mitobim with alternative baiting

  17. Physidae Panpulmonate, non hygrophylid

  18. http://mitos.bioinf.uni-leipzig.de/result.py?hash=xQ93ZHDS

  19. Go to http://mitos.bioinf.uni-leipzig.de/result.py?hash=xQ93ZHDS Look at each tRNA structure prediction. Similar to P. acuta?

  20. http://mitos.bioinf.uni-leipzig.de/result.py?hash=xQ93ZHDS

  21. Go to http://mitos.bioinf.uni-leipzig.de/result.py?hash=xQ93ZHDS Look at each tRNA structure prediction. Similar to P. acuta?

  22. Panpulmonate gastropods, gene order MTgenome Physid S4PE (Illumina) ATP8

  23. Go to http://mitos.bioinf.uni-leipzig.de/result.py?hash=xQ93ZHDS Look at each tRNA structure prediction. Open raw data (ZIP) Obtain (sequence.fas), compare to NC_023253.1 tRNA 11016..11078 /product="tRNA-Leu" /anticodon=(pos:11045..11047,aa:Leu,seq:tag) /codon_recognized="CUN" tRNA complement(12399..12463) /product="tRNA-Leu" /anticodon=(pos:complement(12430..12432),aa:Leu,seq:taa) /codon_recognized="UUR"

  24. gene complement(11150..12268) /gene="CYTB“ /codon_start=1 /transl_table=5 /product="cytochrome b“ /protein_id="YP_008994239.1“ /db_xref="GeneID:18129212"/product="cytochrome b" /protein_id="YP_008994239.1" /db_xref="GeneID:18129212" /translation="MKLKNTVIKEITYLPSPMSLSIWWNMGSIMGLMLFLQILTGFFL SMHYTANIASSFESVIHIIRDVPMGWLVRSLHANGASFFFVFMYLHIARGLYYQSYIT QPKSWSGGTKIFLLSMATAFLGYVLPWGQMSFWGATVITNFLSAIPYVGNIGVKWVWG GFSIGQATLNRFYSLHFILPFIIMILAMLHLVFLHEKGSTNPLGCVNHISKVPFHPYF TWKDSVGFITVLMMLIFVSLYFPNYLGDPENYLLANSMVTPVHIMPEWYFLFAYAILR SIPSKLGGVIALAASIMVLFVLPMIKFEKMISTSFNLMYQVLFWLFCGNFMLLTWLGS CPIEAPYSTLAVPSTFLYFLLLLMMPVSSLLWKYALEK“ tRNA 12277..12332 /product="tRNA-Gly" /anticodon=(pos:12307..12309,aa:Gly,seq:tcc) tRNA 12334..12396 /product="tRNA-His" /anticodon=(pos:12365..12367,aa:His,seq:gtg) tRNA complement(12399..12463) /product="tRNA-Leu" /anticodon=(pos:complement(12430..12432),aa:Leu,seq:taa) /codon_recognized="UUR" gene complement(12505..12618) /gene="ATP8" /db_xref="GeneID:18129213" CDS complement(12505..12618) /gene="ATP8" /codon_start=1 /transl_table=5 /product="ATP synthase F0 subunit 8" /protein_id="YP_008994240.1" /db_xref="GeneID:18129213" /translation="MPQLSPTYGILIFLLVNLTLFFIIILNSYNRNKKITK“ 12241 actgtgaaaa tatgccttgg agaaataata tatttgtttt tagtagtata aatattacac 12301 ttaccttcca agtaaaaaat ctttttaaaa gctcctgtag taggttaaaa taaaccatta 12361 gtttgtgata ctaaaaatat attgtcatct ataggttgtt ttaagaggga cttaacccat 12421 cttagacgct taaagtctat gcattttagt ctgccatctt aaagtaattgtgagtattta 12481 gatttagtat tatggattaa taatttattt cgtaattttt ttatttcgat tatatgaatt 12541 taaaataata ataaagaaca aagtcaagtt tactaatagg aaaataagaa ttccataagt 12601 tggtctgagt tgaggaatgt ttaaatgact acataattgt atctacttaa ttaacagtta

  25. ATCGTGGTAAAACACCGTTATTACTAACATATTAATCAACATCAATGATTTCCGTTCATCCGGCGGGTGTTCATTTTGTAGGCTCCTGGAACATCTTAGACGCTTAAAGTCTATGCATTTTTCTGCCACTACAAATATTAAGAAATTGGTCGTGTTTTATTTATTGATAAGATAACAATAATCGTGGTAAAACACCGTTATTACTAACATATTAATCAACATCAATGATTTCCGTTCATCCGGCGGGTGTTCATTTTGTAGGCTCCTGGAACATCTTAGACGCTTAAAGTCTATGCATTTTTCTGCCACTACAAATATTAAGAAATTGGTCGTGTTTTATTTATTGATAAGATAACAATA Reverse complement (-) strand TATTGTTATCTTATCAATAAATAAAACACGACCAATTTCTTA ATATTTGTAGTGGCAGAAAAATGCATAGACTTTAAGCGTCTAAGATGTTCCAGGAGCCTACAAAATGAACACCCGCCGGATGAACGGAAATCATTGATGTTGATTAATATGTTAGTAATAACGGTGTTTTACCACGAT

  26. http://mitos.bioinf.uni-leipzig.de/result.py?hash=pWBtFmE1 Compare gene order, Find trnS2, check gene predictions Submit to Genbank

More Related