1. The Web frame for NGS output

2. NGS sequencing Tertiary Analysis Secondary Analysis Primary Analysis
Base calling/ Sequence trimming
Secondary Analysis
Assembly or Ref mapping
Tertiary Analysis
Calculate Mapping data/ expression profile
Functional inference

3. Tentative Procedure for RNA –Seq Analysis
No-model Organism
Tentative Procedure for RNA –Seq Analysis QC
Discard the low –confident sequences for 3 groups (three time points)
Program: SolexaQA (
Assembly
Merge all reads from 3 Groups for assembly to form Contigs
Program: Trinity ( 100GB RAM requested
Mapping
Map pair-end reads from each group on Contigs/ Annotate Contigs
Program: LAST ( BLASTx, InterproScan
Expression
Estimate the expression value for each contig in each group (FPKM)
Program: CummeRbund, an R/Bioconductor package (
Functional inference
Functional enrichment analysis in GO and KEGG
Program: Due to no-model organism, we may have to create the mapping identifier in KEGG and GO

4. Tentative Procedure for RNA –Seq Analysis
No-model Organism for Eel transcriptomics
Tentative Procedure for RNA –Seq Analysis QC
Discard the low –confident sequences generated from each library in Hi-seq 200, RNA-seq data, Pairend
Program: SolexaQA (
Assembly
Merge all reads from various libraries for assembly to form Contigs
Program: Trinity ( 100GB RAM requested
Mapping
Map pair-end reads from each group on Contigs/ Annotate Contigs
Program: LAST ( BLASTx, InterproScan
Expression Profiling
Estimate the expression value for each contig in each group (FPKM)
Program: CummeRbund, an R/Bioconductor package (
Functional inference
Functional enrichment analysis in GO and KEGG
Program: Due to no-model organism, we may have to create the mapping identifier in KEGG and GO

5. Tentative Procedure for RNA –Seq Analysis
No-model Organism
Tentative Procedure for RNA –Seq Analysis QC
去除品質較差的定序結果
Program: SolexaQA ( SeqTrim
Assembly
由短序列基因定序結果中，組合出可能的基因表現模組(Merge all reads from 3 Groups for assembly to form Contigs)
Program: Trinity, MIRA, Valvet, etc, multiple CPUs with over 100GB RAM requested
Mapping
以組合出來的長序列基因片段為主體，將短序列歸位到基因主體上(Map pair-end reads from each group on Contigs)
Program: Bowtie, LAST (
Expression
計算與統計不同樣品間同一段基因表現的概況，鑑別出有差異表現基因群(Estimate the expression value for each contig in each group (FPKM))
Program: CummeRbund, an R/Bioconductor package ( rseqC (
Functional inference
將找出的基因群進行功能性分析，找出在不同時間與組織下，與再生機制相關之調控途徑(Functional enrichment analysis in GO and KEGG)
Program: Due to no-model organism, we may have to create the mapping identifier in KEGG and GO
Validation
以Q-PCR來確認與再生相關之基因群表現概況
設計新的實驗來促進或是干擾再生機制，再透過NGS來找出更為精細的調控細節

6. QC by Graphs in SelexaQA

7. Annotations for each Contig
Contig in FASTA (N.A) Translated sequence (AA) in longest ORF
Then perform Sequence Search (BLASTp) on NR, KEGG, GO, pFam (Interpro)

8. Database Structure PK = Contig ID
BLASTx
pFAM
KEGG GO
FPKM
PK = Contig ID
Ref:

9. Query 1: text-based approach
Full –text search on Annotation tables
Sequence Search/ BLAST
Library Compare
Immun
Detail for each contig

10. Query 2 by Sequences BLASTn/ megablast/ tBLASTx Library Compare
Full –text search on Annotation tables
Sequence Search/ BLAST
Library Compare
Worm Contigs
Reference code :

11. Blast Result
Detail for each contig

12. Detail for Each Contig
Interpro/ pFAM

13. Query 3: Library Comparison
Full –text search on Annotation tables
Sequence Search/ BLAST
Library Compare
Dynamic comparison like DDD
Pool A
Submit
Pool B
P-value

14. Table for BLASTX output (DB: NR)
Matched length/Query length
Query_ID
Hit ID
Hit_annotation
Hit_organism
Query coverage
E-value
Contig 1
BAD
elongation factor-1 alpha (EF-1alpha)
Pelodiscus sinensis
97%
0.0
Contig 2

15. Table For KEGG Tables For pFam & GO As the output from each program
#seq_id
hit_seq
alignment_length
identity (%)
e_value
KO_ID
Definition
pathway
Note
comp3_c0_seq1
xla:386604
449
0.84
K03231
elongation factor 1-alpha
ko03013  
RNA transport
ko05134  
Legionellosis
Tables For pFam & GO
As the output from each program
Primary Key

16. The Result in one sheet Contig 1 PF00009/GTP_EFTU PF00010/ xxxxxxxx
Annotation from BLASTx
Results of Pfamscan GO
KEGG_KO
KEGG Pathway
FPKM _cond1
FPKM _cond2
FPKM _cond3
Contig 1
BAD / elongation factor-1 alpha (EF-1alpha) [Pelodiscus sinensis]
PF00009/GTP_EFTU
PF00010/
xxxxxxxx
GO:  GTPase activity  GO:  GTP binding 
K03231/galactose oxidase 
ko Galactose metabolism
190
200 3
Contig 2 -
PF / p450
378 22
1000
Contig 3
CCCC
PPPP
333 45 31

17. Library Compare
0 hr
48 hrs
24 hrs

18. The Way of Redundancy Reduction
Input 700Million reads
500,000 genes
48,000 Genes
Refinement
Final Set
1st Trinity Run
Abundance Sorting
Mapping by BOWTIE2 (LAST?), pick longest one as reduced set