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Today

This midterm assignment focuses on various DNA sequencing and editing techniques such as direct sequencing, precipitate extension products, and editing as needed. Students are required to complete their answers in a word processor, including calculations, figures, and handwritten notes. The assignment also covers topics such as snail and parasite biology, DNA sequencing, gel electrophoresis, primer design, and phylogenetics. Turn-in deadline is Friday, Oct. 19, 24:00h, in the form of a PDF file.

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Today

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  1. Today • HK • Direct Sequening, precipitate • Editing: • *.ppt • MITOS: BLAST, MITOBIM • 15.00h initiate precipitation extension products • Editing as needed

  2. MIDTERM AVAILABLE LIVE WEDNESDAY Oct 10 24.00h Wordfile, *.doc Complete your answers in word processor, insert handwritten calculations, figures as needed Read carefully and answer the full question Use all terms requested to word your answer Calculations, Description concepts, techniques (lecture, class website, on-line, mini-seminars) Edit, analyze sequence data (questions, NCBI, BLAST, Galaxy), Other….. TURN IN AS PDF file FRIDAY Oct 19 24.00h

  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. *.ppt

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

  6. 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

  7. MITOBIM

  8. MITOBIM How to check the output from MITOBIM: Log into Galaxy, Look at MITOBIM output, SELECT ALL And BLASTN against GENBANK https://blast.ncbi.nlm.nih.gov/Blast.cgi

  9. Abstract: About 2000 completely sequenced mitochondrial genomes are available from the NCBI RefSeq data base together with manually curated annotations of their protein-coding genes, rRNAs, and tRNAs. This annotation information, which has accumulated over two decades, has been obtained with a diverse set of computational tools and annotation strategies. Despite all efforts of manual curation it is still plagued by misassignments of reading directions, erroneous gene names, and missing as well as false positive annotations in particular for the RNA genes. Taken together, this causes substantial problems for fully automatic pipelines that aim to use these data comprehensively for studies of animal phylogenetics and the molecular evolution of mitogenomes. The MITOS pipeline is designed to compute a consistent de novo annotation of the mitogenomic sequences. We show that the results of MITOS match RefSeq and MitoZoa in terms of annotation coverage and quality. At the same time we avoid biases, inconsistencies of nomenclature, and typos originating from manual curation strategies. The MITOS pipeline is accessible online at http://mitos.bioinf.uni-leipzig.de.

  10. ASSEMBLY AND ANNOTATION CHALLENGES with NON-MODEL ORGANISMS • tRNAs • Alternative start stop BLAST genome plus cDNA • NC regions, REPETITIVE!

  11. https://www.ncbi.nlm.nih.gov/Taxonomy/Utils/wprintgc.cgi

  12. https://www.ncbi.nlm.nih.gov/nuccore/KT008005.1 Echinostoma paraensei

  13. 9. The Echinoderm and Flatworm Mitochondrial Code (transl_table=9) AAs = FFLLSSSSYY**CCWWLLLLPPPPHHQQRRRRIIIMTTTTNNNKSSSSVVVVAAAADDEEGGGG Starts = ----------**-----------------------M---------------M------------ Base1 = TTTTTTTTTTTTTTTTCCCCCCCCCCCCCCCCAAAAAAAAAAAAAAAAGGGGGGGGGGGGGGGG Base2 = TTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGG Base3 = TCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAG Click here to change format Differences from the Standard Code: Code 9 Standard AAA Asn N Lys K AGA Ser S Arg R AGG Ser S Arg R UGA Trp W Ter * Systematic Range: Asterozoa (starfishes) (Himeno et al., 1987) Echinozoa (sea urchins) (Jacobs et al., 1988; Cantatore et al., 1989) Rhabditophora among the Platyhelminthes (Telford et al. 2000) All parasitic flatworms

  14. CLEAN-UP sequencing reactions. DO NOT DISTURB THE PELLET! These are your sequencing products. 1). Set up two 1.7ml eppendorf tubes, labeled group number, target and F or R and add 100 μl 100% EtOH and 4 μl 3M NaAc. Spin sequencing reactions, then transfer whole volume from the PCR tubes to the correctly labeled tubes. Invert and spin 10’ max RPM at room temperature. 2). Discard supernate, rinse pellet with 400 μl 75% EtOH, spin 5’ max RPM at room temperature. 3). Discard supernate, take out last few drops, dry to air, give to instructor for reading of extension products.

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