1.1. Aim

The aim of this project is to detect genetic determinants related to the development of bat wings by characterizing Transcriptomic and epigenomic data. During the processes of this project, various bioinformatics tools were used and familiarized. It is necessary to mention that the results of this project might differ from the results in the paper because only part of the original data is used.

1.2. Time plan

Day	Hours	Process	Status	Data Type	Software	Estimated Time	DDL
26/3	2	Seminar	Completed	--	--	2h	Seminar
1/4	--	Project Planning	Completed	--	Github	4h	--
6/4	4	Reads quality control	Completed	Illumina	FastQC	3min	Project planning
6/4	4	Reads preprocessing	Completed	Illumina	Trimmomatic	1-10 min/file (2 cores)	Project planning
9/4	4	Genome Assembly	Completed	Illumina	SOAPdenovo	~1.5h (2 cores)	--
9/4	4	Assembly evaluation for SOAPdenovo (no gapcloser)	Completed	Fasta sequences	MUMmerplot	< 5min (1 core)	--
14/4	4	Genome Assembly	Completed	Illumina	SPAdes	~12 h (12 cores)	--
14/4	4	Genome Assembly (after SOAPdenovo)	Completed	Illumina	GapCloser	--	--
15/4	4	Assembly evaluation for spades	Completed	Fasta sequences	MUMmerplot	< 5min (1 core)	Genome Assembly
15/4	4	Assembly evaluation for SOAPdenovo (after gapcloser)	Completed	Fasta sequences	MUMmerplot	< 5min (1 core)	--
15/4	4	Transcriptome assembly	Incompleted	Illumina RNA	Trinity	~1,5h (2 cores)	Genome Assembly
20/4	4	Genome Annotation	Completed	Eukaryotes	BRAKER	?	--
20/4	4	Functional Annotation	Completed	Eukaryotes	EggnogMAPPER	?	--
28/4	4	Calculate heterozygosity of genome	Incompleted	--	BWA	~1.5h (WGS,2cores)	--
29/4	4	RNA mapping	Completed	Eukaryotes RNA	STAR	?	Annotation
29/4	4	Differential Expression	Completed	--	Htseq	< 5min 1 core	--
29/4	4	Differential Expression	Completed	--	DEseq2	?	--
3/5	4	Compare lncRNAs to databases	Incompleted	lncRNA	lncRNAdb, GENCODE lncRNA	1 h	--
3/5	4	Compare homogenic lncRNAs	Incompleted	lncRNA	BLAST	1 h	--
3/5	4	Visualize ChIP-seq data	Incompleted	ChIP-seq data	DiffBind in R	?	--
4/5	4	Deeper analysis of differential expression	Partly Completed	--	hclust, GO analysis	--	RNA mapping
17/5	4	Check; finish wiki	Completed	--	Github	--	--
19/5	4	Check; finish wiki	Completed	--	Github	--	--
24/5	4	Final version of wiki	Incompleted	--	Github	--	Check all

1.3. Data

DNA was extracted from the leg muscle tissue of a single male M. natalensis. RNA was extracted from paired forelimbs and hindlimbs from three individuals (biological replicates) at three developmental stages (CS15, CS16, and CS17). The subset of original data is provided through UPPMAX under /proj/g2021012/2_Eckalbar_2016.

There are 4 datasets which are sel1, sel2, sel3, and sel4 in this folder. Each dataset contains data from whole-genome sequencing, ChIP-sequencing, and RNA-sequencing. The additional folder contains the reference genome assemblies for each subset of data.

Data	Tissue	Stage	Tool
WGS	Leg muscle of a single M. natalensis male	Adult	Illumina
ChIP-seq	Bat forelimb and hindlimb	CS15, CS16, CS17	Illumina
RNA-seq	Bat forelimb and hindlimb	CS15, CS16, CS17	Illumina

WGS Data

The data in the following table is used for DNA assembly (SOAPdenovo).

Read	Average insert Library size (bp)
SRR5819794	400
SRR5819795	800
SRR5819796	9000
SRR5819797	175
SRR5819798	3000
SRR5819799	5500

RNA Data

ID	Tissue	Development stage
SRR1719013	Forelimb	CS15
SRR1719014	Forelimb	CS15
SRR1719015	Forelimb	CS15
SRR1719016	Hindlimb	CS15
SRR1719017	Hindlimb	CS15
SRR1719018	Hindlimb	CS15
SRR1719204	Forelimb	CS16
SRR1719206	Forelimb	CS16
SRR1719207	Forelimb	CS16
SRR1719212	Hindlimb	CS16
SRR1719214	Hindlimb	CS16
SRR1719242	Hindlimb	CS16
SRR1719208	Forelimb	CS17
SRR1719209	Forelimb	CS17
SRR1719211	Forelimb	CS17
SRR1719213	Hindlimb	CS17
SRR1719241	Hindlimb	CS17
SRR1719266	Hindlimb	CS17

These datasets including paired and unpaired reads.

Data Structure

The data and scripts of this project are stores in the following structure. The raw data is in the 2_Eckalbar_2016 and kept unchanged during the project. The unziped_data folder stores the data which has been copied and unzipped from the raw data folder. All scripts are in the script folder and all results are saved in the result folder.

.
├── 2_Eckalbar_2016 -> /proj/g2021012/2_Eckalbar_2016/
├── result
│   ├── 1_quality_control
│   ├── 2_trimming
│   ├── 3_dna_assembly
│   ├── 4_assembly_validation
│   ├── 5_transcriptome_assembly
│   ├── 6_repeatmasker
│   ├── 6_softmask_test
│   ├── 7_ref_mapping
│   ├── 7_rna_mapping
│   ├── 8_braker_annotation
│   ├── 8_ref_annotation
│   ├── 8_ref_eggnog
│   ├── 9_htseq
│   └── test
├── script
│   ├── 1_fastqc_chip.sh
│   ├── 1_fastqc_rna_raw_trimmed.sh
│   ├── 1_fastqc_rna.sh
│   ├── 1_fastqc_rna_trim.sh
│   ├── 1_fastqc_wgs.sh
│   ├── 2_trim_rna.sh
│   ├── 3_GapCloser_SOAP_wgs.sh
│   ├── 3_SOAPdenovo_wgs.sh
│   ├── 3_spades_assembly_wgs.sh
│   ├── 4_MUMmer_soapdenovo_nogapcloser_wgs.sh
│   ├── 4_MUMmer_soapdenovo_wgs.sh
│   ├── 4_MUMmer_spades_wgs.sh
│   ├── 5_trinity_rna_pair.sh
│   ├── 5_trinity_rna_single.sh
│   ├── 6_hard_repeatmasker.sh
│   ├── 6_repeatmasker_wgs.sh
│   ├── 6_repeatscout_wgs_filter.sh
│   ├── 6_repeatscout_wgs.sh
│   ├── 6_soft_repeatmasker.sh
│   ├── 7_ref_mapping.sh
│   ├── 7_rna_mapping.sh
│   ├── 7_test.sh
│   ├── 8_annotation_new.sh
│   ├── 8_BRAKER_annotation.sh
│   ├── 8_genemark.sh
│   ├── 8_ref_annotation.sh
│   ├── 8_ref_eggnog.sh
│   ├── 8_test.sh
│   ├── 9_htseq_count.sh
│   ├── slurm_log
│   ├── soapdenovo_config.txt
│   └── soapdenovo.log
└── unziped_data
    ├── GCF_001595765.1_Mnat.v1_genomic.gff
    ├── NW_015504249.1.gff
    ├── quast_output
    ├── sel2_NW_015504334.fna
    └── sel3_NW_015504249.fna

21 directories, 38 files

1.4 Workflow