1	Denovo genome assembly and analysis
2	outline De novo genome assembly Gene finding from assembled contigs Gene annotation
3	Denovo genome assembly
4	Gene finding To find out coding region on genome sequence
5	Gene Annotation For each gene…. Conserved? Domain? Function?
6	get reads file download a random generated reads file http://163.25.92.61/course/randomreads30k.fasta open CLC to assemble contigs from reads
7	NGS import the reads file
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10	Denovo assembly
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13	report
14	assembled contigs
15	export fasta file
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17	Glimmer Glimmer is a system for finding genes in microbial DNA, especially the genomes of bacteria, archaea, and viruses. (Gene Locator and Interpolated Markov ModelER) http://www.cbcb.umd.edu/software/glimmer/ Center for Bioinformatics & Computational Biology, University of Maryland Paper about Glimmer 1.0 S. Salzberg, A. Delcher, S. Kasif, and O. White. Microbial gene identification using interpolated Markov models, Nucleic Acids Research 26:2 (1998), 544-548. Glimmer2.0 A.L. Delcher, D. Harmon, S. Kasif, O. White, and S.L. Salzberg. Improved microbial gene identification with GLIMMER, Nucleic Acids Research 27:23 (1999), 4636-4641. Glimmer 3.0 A.L. Delcher, K.A. Bratke, E.C. Powers, and S.L. Salzberg. Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics 23:6 (2007), 673-679.
18	http://www.cbcb.umd.edu/software/glimmer/
19	Or download glimmer from here wget http://163.25.92.61/course/glimmer302.tar.gz
20	Glimmer install extract tar zxvf glimmer302.tar.gz tree -d glimmer3.02/ go into directory of glimmer’s source code cd glimmer3.02/src/ pwd compile the binary code make executable binary will be located in ( glimmer3.02/bin/ )
21	Concept of glimmer Trainning model from… Known genes Genes from evolutionary relative organism Open reading frames
22	4 steps to run the glimmer long-orfs This program identifies long, non-overlapping open reading frames (orfs) in a DNA sequence file. extract This program reads a genome sequence and a list of coordinates for it and outputs a multifasta file of the regions specified by the coordinates build-icm This program constructs an interpolated context model (ICM) from an input set of sequences. glimmer3
23	g3-from-scartch.csh glimmer3.02/scripts/ g3-from-scratch.csh genome.fasta mygenome The script would then run the commands: long-orfs -n -t 1.15 genome.fasta mygenome.longorfs extract -t genome.fasta mygenome.longorfs > mygenome.train build-icm -r mygenome.icm < mygenome.train glimmer3 -o50 -g110 -t30 genom.seq mygenome.icm mygenome
24	Output of glimmer (xxx.predict) >gi\|15638995\|ref\|NC_000919.1\| Treponema pallidum subsp. pallidum str. Nichols, complete genome orf00001 4 1398 +1 6.22 orf00003 1641 2756 +3 2.89 orf00004 2776 3834 +1 5.47 orf00005 3863 4264 +2 2.77 orf00006 4391 6832 +2 7.08 orf00007 6832 7074 +1 0.25 orf00008 7317 7967 +3 6.92 orf00009 7997 8260 +2 2.91 orf00010 9515 8340 -3 2.80 orf00011 9838 9984 +1 0.10 orf00013 10237 10362 +1 6.02 orf00014 10396 12378 +1 3.77 orf00015 12545 13210 +2 8.04
25	Modification of the script g3-from-scartch.csh vi ../scripts/g3-from-scartch.csh set awkpath = /fs/szgenefinding/Glimmer3/scripts set glimmerpath = /fs/szgenefinding/Glimmer3/bin set awkpath = ~/glimmer3.02/scripts set glimmerpath = ~/glimmer3.02/bin
26	vi 編輯器: vi filename w 儲存 q 離開vi wq 儲存後離開 q! 不儲存就離開
27	Convert coordinate file into fasta format (single fasta file) extract Usage: extract genome_file coord_file > fasta_file
28	for multiple fasta file coordinate convert use home-made script to re-format coordinate file http://163.25.92.61/course/multipredict.pl multi-extract Usage: multi-extract genome_file coord_file > fasta_file
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30	NetBlast The BLAST client, or blastcl3, bypasses the web browser and interacts directly with the NCBI BLAST server that powers the NCBI web BLAST service ftp://ftp.ncbi.nlm.nih.gov/blast/executables/release/LATEST/ But you can download here… cd ~ (go back to your home directory) wget http://163.25.92.61/course/netblast-2.2.25-ia32-linux.tar.gz extract tar zxvf netblast-2.2.20-ia32-linux.tar.gz
31	blastcl3 netblast-2.2.25/bin/ ./blastcl3 -p program -i input_sequence -d dbname -o output_file -p (blastn, blastx, blastp, tbastn tblastx) -i (query file, predice genes here) -d (database name) nr, NCBI non-redundant database -o (output file)
32	Blast programs
33	"./blastcl3" ./blastcl3 -p blastn -i mygene.fasta -d nt -o mygeneblast.html -m 2 -K 1 -T T