De Novo Assembly (Pseudomonas aeruginosa MAPO1 ) Sample to Insight

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1 De Novo Assembly (Pseudomonas aeruginosa MAPO1 ) Sample to Insight 1

2 Workflow Import NGS raw data QC on reads De novo assembly Trim reads Finding Genes BLAST Sample to Insight

3 Case Study Pseudomonas aeruginosa MAPO1 variant re-sequencing Olivas AD et al., PLoS One, 2012 SRP SRX / SRR Single reads SRX / SRR mate-pair (distance: ) SRX114600/ SRR paired-end (distance: ) Sample to Insight

4 Demo Dataset Please unzip the file after you download from CLC Bio website Sample to Insight

5 Import NGS raw data Import Single Reads File 1. Select Single_read.fastq file 2. Uncheck all items in the General options 3. Confirm the quality score is NCBI/Sanger or Illumina pipeline 1.8 Sample to Insight

6 Import Mate-Paired Data Select Mate_pair_1.fastq and Mate_pair_2.fastq files Check-on Paired reads in general option Select Mate-pair in Paired reads information Set Max distance = 3800 Set Min distance = 2000 Sample to Insight

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9 Select the location to save the mate-pair reads Press Finish

10 Import Paired-end Data Check-on Paired reads in general option Select Paired-end in Paired reads information Set Max distance = 350 Set Min distance = 150 Select 2 files: Sample to Insight

11 Please create a new folder and organize your data list

12 QC on reads NGS Core Tools Create Sequencing QC Report Sample to Insight

13 About QC on reads Please confirm uncheck discard quality score when you import reads Process analysis file by file (you can use batch function) The quality score in CLC GWB is transformed to PHRED score Sample to Insight

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15 Create report

16 Check-on items, save result

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19 Please repeat the procedure to get the reads QC report for mate-paired reads and paired-end reads Sample to Insight

20 Trim reads NGS Core Tools Trim Sequences Sample to Insight

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22 Set p value = 0.05 (default) Next Set discard reads below length = 15 N Sample to Insight

23 Check on Save broken pairs Save the result Next

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26 De novo assembly De Novo Sequencing De Novo Assembly Sample to Insight

27 Uncheck Automatic word size, set word size = 45 Uncheck Automatic Bubble size, set bubble size = 9 Set min contig length = 1000 Sample to Insight

28 De Brujin Grpah for De novo Assembly *Word size = k-mer size e.g k=16 Sample to Insight

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31 Bubble or sequencing systematic error

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35 Scaffolding

36 Deployment for De Novo Assembly Fast mode : De novo contig sequences only Slow Mode : Take de novo assembled contigs as reference template, then use all reads to process reference mapping (re-mapping) + update contigs Sample to Insight

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38 Check-on Create report, save the result Next Assign the location to save contigs Sample to Insight

39 BLAST For BLAST Extract consensus sequence of contigs Process BLAST Sample to Insight

40 Extract consensus sequences Open de novo contig table Sample to Insight

41 Select all contigs, press Extract Contigs

42 Process BLAST

43 Select the consensus sequence Next

44 Select query for Bacteria

45 Save the result, assign the location Press Finish

46 BLAST result Sample to Insight

47 Finding Genes Classical Sequence Analysis Nucleotide analysis find Ope Sample to Insight

48 Select extracted de novo contigs

49 Set minimum length of OR Assign start codon: AUG, C Sample to Insight

50 Create annotated sequence and save the result

51 Assign path to save the ORF finding result

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53 Extract ORF sequence Go to Plug-ins Download Plug-ins Select Extract Annotations Press Download and Install Press close and restart the software Sample to Insight

54 Classical Sequence Analysis General Sequence Analysis Sample to Insight

55 Select annotated ORF sequences

56 Select Type as ORF

57 Assign saving path Finish

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59 For more information Please welcome to Sample to Insight