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Paper 5: Durian(Durio zibethinus) genome analyses

Zhiwei LI

1. Aim of the project.

  1. De novo assembly of Durian and do structural and functional annotation.
  2. Transcriptome assembly and analyses differential expression.
  3. Biological interpretation of the results.
  4. Further analyses.

2. Type of analyses

Check data description

Table of analyses and the employed application.

No reads quality control and reads preprocessing since we are working with PacBio reads.

Analysis Type Software Installed/codes ERT Input/note
1.DNA assembly Assembly Canu UPPMAX/1_gene_assembly.sh ~17h (4 cores) PacBio reads;
2.Mapping illlumia reads against assembly Mapping/Aligner BWA( BWA-MEM) UPPMAX//4_MappingIllumina.sh ~ 1h Illumina read to PacBio assembly (for BAM file as Pilon input)
3. DNA assembly Improvement Assembly Improvement Pilon UPPMAX/7_Pilon.sh ~30min PacBio assembly; BAM file;
4. Assembly evaluation ( must do after 3(Pilon), optional for 1(Canu)) Fasta sequences QUAST Locally/3_PacBio_AssemblyQC.sh ~ Assembly
RNA raw data quality check and trim and check again after trim FastQC Trimmomatic UPPMAX/2_RNA_rawData_QC.sh ~
5. Mapping (Aligner) Eukaryotic RNA Tophat UPPMAX/5_tophatFolder/ ~5h (2 cores) Downloaded DNA sequence, different pairs of RNA reads. Part for 6, all for 10.
6. RNA assembly Illumina RNA Trinity(need map../no map) UPPMAX/9_trinity_withBAM.sh ~5.5h (4 cores) Merged BAM file from Tophat.
7. Functional annotation Eukaryotes EggNOGmapper Online/Submitted online output maker.protein.fasta from Maker2
8. Find relatedness proteins FASTA Not provided Download online: (arabidopsis) AND "Arabidopsis thaliana" in: NCBI protein database - Reduce the number of species to run faster (used arabidopsis)
9. Two iterations Annotation (structural)!!!first structural then functional!!! Eukaryotes Maker2 UPPMAX/11_Maker2/ Two iterations 6,12h(long) (4 cores) (Input: assembly trinity output and relatedness protein)
10. Read counting Count features HTSeq UPPMAX/12_forEachPair_HTSeq/ ~ BAM files from Tophat, gff from Maker2
11.Differential Expression Comparison (input: HTSeq) DESeq2 (R library) Locally, UPPMAX/15_DEseq.R Variable counts from HTseq
12. Visualization of the genome Reads and genomic annotation IGV/Artemis Locally Variable genome, .Gff, bam(one of each, then add more)

Basic analyses:

● Genome assembly of PacBio reads.

● Correct the assembly with Illumina reads.

● Assembly quality assessment.

● Structural and functional annotation.

● Transcriptome assembly.

● Differential expression analyses.

● Biological interpretation of the results.

Extra analyses:

● Assembly with different parameters.

● Assembly evaluation with more than one method.

● Deeper analyses of differential expression analyses: e.g. different comparisons.

Time bottlenecks

The biggest bottleneck will be DNA assembly of PacBio reads(17h). RNA assembly, annotation and mapping will take more than 5h.

3. Time frame

Check work log

4. Data handling

There are three types of data I have: RNA raw data, RNA trimmed data and WGS trimmed data. Thay are all fastq files.