Introduc)on to annota)on with Artemis. Download presenta.on and data

Transcription

1 Introduc)on to annota)on with Artemis Download presenta.on and data

2 Annota)on Assign an informa)on to genomic sequences????

3 Genome annota)on 1. Iden.fying genomic elements by: Predic)on (structural annota.on of ORF, gene structure, coding regions,...) i.e. ab ini&o predic.on (gene finder) Similarity (known elements) i.e. BLAST 2. Assign a biological informa.on to these elements Func.onal annota.on such as func.on, expression, regula.on, diversity... How? Automa.c Pipeline for genome scale annota.on Tools (web services) such as Maker: hop:// lab.org/sorware/maker.html + Fast, only computer analysis - high error rate, do not find unexpected features Manual annota.on by human exper.se + Very good quality - slow, different annota.on between different annotators

4 Annota)on Process Raw DNA sequence RNAseq, ESTs, proteins, genes... Gene Finder Repeat Finders DoOer BlastN trna scan BlastX Manual cura.on Repeats Transcript trna Pseudo- genes Genes

5 Annota)on Process Raw DNA sequence RNAseq, ESTs, proteins, genes... Gene Finder Repeat Finders DoOer BlastN trna scan BlastX Manual cura.on Repeats Transcript trna Pseudo- genes Genes DoOer BLASTP PFam Prosite SignalP Psort Manual cura.on

6 Artemis: Genome browser and Annota)on tool Can be downloaded at hop:// Current version 15.0

7 Artemis: Genome browser and Annota)on tool What is Artemis? Artemis is a DNA sequence viewer and annota.on tool that allows visualisa.on of sequence features and the results of analyses within the context of the sequence, and its six- frame transla.on. Artemis is wrioen in Java, reads EMBL or GENBANK format sequences and feature tables, and can work on sequences of any size. On Unix and GNU/Linux systems, given an EMBL accession number Artemis also can read an entry directly from the EBI using Dbfetch. Read An Entry This func.on only reads the feature sec.on of the input file Artemis can read these feature files format: EMBL & GenBank GFF v3 FASTA files output of MSPcrunch output of blastall (must be run with the m 8 flag) or blast+ (- oudmt 6) Mapping alignment file (BAM/VCF) Launch Artemis In a Linux terminal type art or artemis

8 Entry: The input file Open an already annotated sequence (EMBL/GenBank features format) In Annota.on- 1 directory, open EU gb Open an annota.on - > Ctrl E

9 Select: Select an annota)on or a base range Select - > Feature selector...

10 View: view sequences and annota)ons View- > overview View- > base of selec.on as Fasta/amino- acid of selec.on as Fasta (Features or user selec.on)

11 Goto: search for sequences (paqern) and annota)ons Goto- > Navigator Test the base paoern! (i.e Look for the AAAAAAAAAAAAAAAAAAAA paoern in the BAC sequence)

12 Edit: Edit and changes annota)ons Edit- > Ctrl E Change: Keys, Qualifier, Loca.on, strand,...- > test by changing any features! Edit- > Selected feature (s) - > duplicate, remove, delete, merge... features Bases- > Reverse & complement, delete and add bases to the sequence

13 Create: create annota)ons Create- > New feature; Feature from base range Mark ORF: create a new entry with all ORF in the current sequence Mark from paoern Mark ambigui.es

14 Run: Run analysis tools Select an annota)on and RUN external programs on UNIX WEB tools : BLAST (NCBI), pfam, SMART (single modular architecture research tool) Need to be installed on a Linux Computer: BLAST ( blastp, tblastn, etc), EMBOSS (sigcleave, pepstats, helixturnhelix), Clustalx, Jalview,...

15 Run: Run analysis tools Select an annota)on and RUN external programs on UNIX WEB tools : BLAST (NCBI), pfam, SMART (single modular architecture research tool) Need to be installed on a Linux Computer: BLAST ( blastp, tblastn, etc), EMBOSS (sigcleave, pepstats, helixturnhelix), Clustalx, Jalview,... To be configured!

16 Graph: Display graph gc content,...

17 Display third party analysis Entry with an embl, genbank, gff or tabular format i.e Artemis may display BLAST results in tabular format (flag blastall m 8, BLAST+ - oudmt 6) Open different blast results in tabular format (*.bln and *.blx) and Fgenesh gene predic.on File- > Read an entry- > In Annota.on- 1/BLAST/ and Annota.on- 1/FGENESH Open Fgenesh (gene finder) results in Gff format

18 Read BAM/VCF (BAM binary Sam = file containing alignment data) See SAM Tools web site: hop://samtools.sourceforge.net BAM files need to be sorted and indexed using SAMtools : samtools sort <in.bam> <out.prefix> samtools index <sorted.bam> Ex. Mapping of 100 M reads from C. canephora cdna on the BAC seq. using Mosaik Load the *.sorted.bam using the Read BAM op.on: Try all reads and mm1 reads File - > Read BAM/VCF - > select MAPPING - > *.sorted. bam (alignment with 1 mm) or File - > Read BAM/VCF - > select MAPPING/ALL- mixed - > *.sorted. bam

19 Read BAM/VCF (BAM binary /Sam = files containing alignment data) See SAM Tools web site: hop://samtools.sourceforge.net VCF (Variant Call Format) files need to be sorted and indexed using bcrools The VCF files need to be compressed and indexed using bgzip and tabix respec.vely Open in the Annota.on- 1 directory any *.vcf.gz files (Leaf, Root, Stamen, Pis.l) File - > Read BAM/VCF - > select Annota.on- 1/VCF - > *.sorted. bam DELETION C A T MULTIPLE G INSERTION STAMEN ROOT PISTIL LEAF

20 Read BAM/VCF (BAM binary Sam = file containing alignment data) Correc)on of annota)on with RNAseq: - Exon/intron boundaries - Valida.on of exons - Discovery of new exons - new coding and non coding genes - TE

21 Read BAM/VCF (BAM binary Sam = file containing alignment data) Correc)on of annota)on with RNAseq: - Exon/intron boundaries - > splice site mapping (Alignment with MapSplice) File - > Read BAM/VCF - > select MAPSPLICE - > *.sorted. bam

22 Read BAM/VCF (BAM binary Sam = file containing alignment data) Correc)on of annota)on with RNAseq: - Exon/intron boundaries - > splice site mapping

23 Read BAM/VCF (BAM binary Sam = file containing alignment data) Load several *.sorted.bam using the Read BAM op.on according to organes/.ssus. Graph- > coverage LEAF 40 M reads ROOT 30 M reads PISTIL 26 M reads STAMEN 20 M reads

24 Read BAM/VCF (BAM binary Sam = file containing alignment data) Load several *.sorted.bam using the Read BAM op.on according to organs/.ssues. LEAF 40 M reads ROOT 30 M reads PISTIL 26 M reads STAMEN 20 M reads

25 Now You re ready to annotate another BAC clone Open a FASTA sequence Select the file Annota.on- 2/BAC2.fasta Save the sequence as annota.on file in EMBL Format: BAC2.embl Close current file and open Annota.on file with Artemis (BAC2.embl)

26 Now You re ready to annotate another BAC clone You need predic)on of coding regions: Load Fgenesh predic.ons done at hop://linux1.sorberry.com/berry.phtml - > gff Load Augustus predic.ons done at hop://bioinf.uni- greifswald.de/augustus/ Load genemark predic.ons done at hop:// lab.org/sorware/maker.html Blast results: Load BLAST+ results vs trembl and/or swissprot All predic.ons can be done at MAKER web site access to the web annota&on service (as register user or Guest account)

27 Load analysis Load gene predic)ons: Fgenesh.gff, augustus.gff, genemark.gff File - > Read an entry - > select FGENESH or MAKER directories and *.gff files Fgenesh augustus genemark

28 Load analysis- > Layers Evidences suppor)ng predic)ons Load Blast(n) vs EST, blast vs proteins (Swissprot, Trembl,..), blast vs specialized db (transposable elements, REPBASE..) File - > Read an entry - > select BLAST dir. and any *.bln and *.blx blast results Fgenesh EST C. canephora augustus genemark TREMBL EST C. arabica

29 Load analysis- > Layers Evidences suppor)ng predic)ons Blast(n) vs EST (C. canephora, C. arabica), blast vs proteins (Swissprot, Trembl,..), blast vs specialized db (transposable elements, REPBASE..) File - > Read BAM/VCF - > select MAPPING - > *.sorted. bam (alignment with 1 mm)

30 Validate and Edit an annota)on Select a correct annota.on from the layer (i.e. Fgenesh) Edit - > move selected feature to BAC2.embl Then Ctrl E to edit annota.on in the layer of Annota.on (i.e. change coordinates, change keys or add qualifier,...)

31 Create an annota)on (ctrl E) Select a key for the annota.on i.e gene or CDS or repeat_region Type a loca.on Select an orienta.on (reverse frame)

32 Artemis: Genome browser and Annota)on tool Create an annota)on Create - > Mark ambigui.es (Gaps, Ns)

33 Artemis: Genome browser and Annota)on tool Create an annota)on Create - > Mark ambigui.es (Gaps, Ns), mark ORF,...

34 Now try to annotate a gene!