1. DAY 1. GENERAL ASPECTS FOR GENETIC MAP CONSTRUCTION SANGREA SHIM

2. INDEX  Day 1  General aspects for genetic map construction  Genetic polymorphism and recombination frequency  Genotyping using molecular marker  Map construction (phenotype, AFLP, RFLP)  Sequencing method  Next generation sequencing  Whole genome reference sequence  Reference sequencing for Genotyping  Retrieving sequence polymorphism  Genetic map construction (SNP, InDel)

3. GENETIC POLYMORPHISM & RECOMBINATION FREQUENCY

4. GENOTYPING USING MOLECULAR MARKER An Integrated High-density Linkage Map of Soybean with RFLP, SSR, STS, and AFLP Markers Using A Single F2 Population Xia et al. 2008

5. MAP CONSTRUCTION An Integrated High-density Linkage Map of Soybean with RFLP, SSR, STS, and AFLP Markers Using A Single F2 Population Xia et al. 2008

6. NEXT GENERATION SEQUENCING  Sequencing  Sanger’s Dideoxy Termination  Using dNTPs  Electrophoresis in capillary gel  Read dye colors one-by-one  Average 700~900 bp  Massive Parallel Sequencing Platform  So called Next Generation Sequencing platform  SOLiD (Sequencing by Ligation), Illumina (Sequencing by synthesis), 454 (Pyrosequencing)  Read 50+35(50+50), 50~300, 700 bp  1200~1300, ~3000, 1 million reads per run

7. NEXT GENERATION SEQUENCING Sequencing technologies – the next generation Michael et al. Nature review genetics 2010

8. WHOLE GENOME REFERENCE SEQUENCE  Polymorphism discovered by comparison  Reference is required for comparison  So, the reference genome is obligated  Making contigs which is constituted by unique sequences combination using PE or small size MP  Scaffolding which includes less unique sequences (i.e. repetitive sequences) using large insert size MP library sequences  Anchor the scaffold using genetic map  But, genetic map constituted by several types of molecular marker is not able to translate to sequence information

9. RESEQUENCING FOR GENOTYPING  GET Polymorphism!, Treat it as a marker or locus!  SNPs  Small size InDels  Align several depth of raw read sequence against Ref.  Statistics  Lots of alignment software is available  BLAST, BLAT, BWA, BOWTIE-series…..  Aligner which use BWT as a main algorithm are famous  Fast, efficient

10. RESEQUENCING FLOW CHART SolexaQA bwa bowtie2 bwa bowtie2 Alignment samtools SAM samtools BAM Sorted BAM samtools bcftools samtools bcftools pileup VCF selection JoinMap4 Map construction DNA/RNA NGS platform Raw read Sequences Raw read Sequences Quality trimming

11. RETRIEVING SEQUENCE POLYMORPHISM  BOWTIE2 or BWA are just align the bulky reads to reference sequence  Making SAM(sequence alignment/mapping)/BAM(binary sequence alignment/mapping) as a result  Several types of statistics or inferences can be adapted to retrieving polymorphism (Picard, GATK)  Samtools package is used in retrieving variants  The VCF(variant calling format) is the ouput file

12. GENETIC MAP CONSTRUCTION Selection of a core set of RILs from Forrest x Williams 82 to develop a framework map in soybean Wu et al. 2011

13. HURDLES ON THE ROAD TO GENETIC MAP  Output of calling variation is a VCF format  JoinMap input file is LOC format  Is there a Converter between the VCF and LOC?  Make converter program, Make genetic map yourself  These are the final goal of this courses

14. TODAY’S PRACTICE  Make a connection to remote computer  Get used to Linux system  Get familiar with python2.7

15. THANK YOU  If you have a question, please ask me.

16. DAY 1. PRACTICE- BASIC LINUX COMMAND TAEYOUNG LEE

17. CONNECTING  Server is located in Seoul National University campus  Connect to server computer using putty SSH client program  Download at http://www.chiark.greenend.org.uk/~sgtatham/putty/download.htmlhttp://www.chiark.greenend.org.uk/~sgtatham/putty/download.html

18. CONNECTING  Execute putty  Put IP address (147.46.250.193) at Host Name and click OPEN

19. CONNECTING  ID : trainee  PW : bogor  Then you are in server now  Only white character on black background

20.  ls  Listing files and directories  cd  Change directory  Practice) enter into /data2/python BASIC COMMAND IN LINUX

21.  mkdir  Make directory  Usage) mkdir dir_name  Practice) make directory named as your name BASIC COMMAND IN LINUX

22.  vi  Open text editing program  Make new text file  usage) vi filename_to_edit vi filename_to_make  Practice) make text file named as yourname in your directory, write something and save it  Insert, replace, esc  :q :w :wq :q! BASIC COMMAND IN LINUX

23.  mv  Moving files or directories  Rename files or directories  Usage) mv present_file_path file_path_to_move  Practice)  Change directory into upper directory  cm) cd..  Make some text file by vi  Move text file to your directory  Rename text file BASIC COMMAND IN LINUX

24.  cp  Coping files or directories  Usage) cp file_path file_path_to_copy  cp can rename file  If you want to copy directory, you have to use –r option  Cp –r dir_path dir_path_to_copy  Practice)  Make directory in your directory  Copy some file into directory with rename and w/o rename BASIC COMMAND IN LINUX

25.  rm  Removing files or directories  Usage) rm file_name  If you want to remove directory, you have to use –r option  rm –r dir_name  Practice)  Remove the directory and file BASIC COMMAND IN LINUX

26.  less  Read only text viewer  Have advantage for large size text file  Usage) less file_name  Searching function  /  Practice)  Open large text file by vi and less  /data2/python/Gmax_109_gene_exons.gff3  Use searching function  /Gm12 BASIC COMMAND IN LINUX

27.  wget ftp://ftp.arabidopsis.org/ home/tair/Sequences/whole_chromosomes/tai r9_Assembly_gaps.gff

28.  cat  Concatenate files  Print out files  Usage cat file_name1 file_name2 …  Practice)  Print out file by cat  Print out file three times BASIC COMMAND IN LINUX

29.  grep  Grep the lines contain some words  Usually use with cat  Usage) cat file_name | grep ‘word’  ‘|’ mean after  This usage mean we grep line which contain some word after print out file  Various useful options  -v : vanish  -c : count  ‘word1\|word2’ = word1 or word2  grep ‘word1’ | grep ‘word2’ = word1 and word2  Practice)  Grep ‘Gm12’ in /data2/python/Gmax_109_gene_exons.gff3  Grep ‘Gm12’ or ‘Gm15’ in same file  Grep ‘gene’ and ‘mRNA’  Count line contain ‘Gm12’  Vanish line contain exon or CDS or mRNA BASIC COMMAND IN LINUX

30.  sort  Sorting file  Usually use with cat  Usage) cat file_name | sort  Various useful options  -k sort by column  -u sort and remove redundancy  -n numeric sort  -r reverse  -d delimiter setting  Practice)  Sort /data2/python/Gmax_109_gene_exons.gff3 by start position(by column and numeric) BASIC COMMAND IN LINUX

31.  cut  Cutting column in file  Usually use with cat  Usage) cat file_name | cut –f n (n : integer)  Practice)  Retrieve chromosome, start position, end position in /data2/python_study/Gmax_109_gene_exons.gff3 BASIC COMMAND IN LINUX

32.  >  Standard input, output vs. file input, output  Input and output on screen or file  > can save standard output to file output  cat file_name | grep ‘word’ > output_file  >>  >> also can save standard output to file output  But just adding! BASIC COMMAND IN LINUX

33.  Fasta file  /data2/python/ap2.fa  Fastq file  /data2/python/example.fastq  Gff file  /data2/python/Gmax_109_gene_exons.gff3  Python file!  /data2/python/1stday.py HANDLE FILE

34.  Make a new text file named as new.txt  The file contain  Gm01,1,23  Gm04,4,56  Gm03,6,78  Gm04,8,10  Copy new.txt into new.copy  Remove new.copy  Using cat, print the contents of new.txt  Using grep, print the contents the new.txt contain Gm04  Using cut, print the first column of new.txt and save it as a file named as new.txt.cut

35. THAT’S IT FOR TODAY  Q & A