replicating transcriptomic analysis before DE

[rx32940]

General description of the project:

• MinION sequencing
• maintenance host vs. incidental hosts
• comparative study

[rx32940]

• replicate Alejandro's results on the first trial sequencing data:
  • [x] quality check
  • [x] map to Lepto reference genome (minimap2)
    • Leptospira_interrogans_Copenhageni_CI & CII (AE016823 & AE016824)
  • [ ] map to host reference genome

[rx32940]

replicate Alejandro's results on the first trial sequencing data:

• [x] quality check

• [ ] map to Lepto reference genome (minimap2)

  • Leptospira_interrogans_Copenhageni_CI & CII (AE016823 & AE016824)
• [ ] map to host reference genome

for quality check, only barcode01 was done. the quality of the sequence is very bad. for the purpose of replication. Moving to mapping now.

• I had concatenate all runs in barcode01 into a single fastq file when doing the quality check. https://github.com/rx32940/Lepto-Minion/blob/master/fastqc_sapelo2.sh see the results in: /scratch/rx32940/minion_blood_simulation/test_runs/QC

[rx32940]

replicate Alejandro's results on the first trial sequencing data:

• [x] quality check

• [x] map to Lepto reference genome (minimap2)

  • Leptospira_interrogans_Copenhageni_CI & CII (AE016823 & AE016824)
• [x] map to host reference genome

mapped all barcodes to the Leptospira reference genome. The percentage of mapping is very close to that of Alejandro. Difference probably caused by different reference genomes were used.

• around half % mapped to lepto reference genome with Bovine blood + Lepto sample (ex. barcode01), close to 100% for EMJH media + Lepto sample (ex.barcode02)
• [ ] need to ask Alejandro about the specific accession of the reference genome
• [ ] have Alejandro done mapping for rest of the barcode samples (07-11)

mapping script on sapelo2 with minimap2: https://github.com/rx32940/Lepto-Minion/blob/master/minimap2_sapelo2.sh see the results in: /scratch/rx32940/minion_blood_simulation/test_runs/map

questions for next step:

• [x] what reference genome to use for bovine?
• [x] how to map mammal genome?

[rx32940]

replicate Alejandro's results on the first trial sequencing data:

• [x] quality check

• [x] map to Lepto reference genome (minimap2)

  • Leptospira_interrogans_Copenhageni_CI & CII (AE016823 & AE016824)
• [x] map to host reference genome

questions for next step:

• [x] what reference genome to use for bovine?
• [x] how to map mammal genome?

reference genome for bovine (Bos taurus (cattle)) : assembly ARS-UCD1.2

Alejandro's mapping result: (I probably used different reference genome for cattle and for Leptospira_interrogans_Copenhageni)

• [ ] summary table for my own mapping stats

[rx32940]

• new data received, can be found at:

  /project/lslab/lab_shared/LeptoTranscriptome/PolyA_Experiment

• Barcode01, Barcode02, and Barcode 12 are misidentified barcode sequences

• unclassified folder is the samples which barcode were failed to be classified by the barcoder

• some Leptospira isolates treated with PolyA tailing and without PolyA tailing. The library preparation sequencing kit from Nanopore Technologies only targeted the PolyA tailed samples cDNA samples. I think it is only about 2% of the total RNA from the Leptospira are PolyA tailed (Dr. Sree can correct me for this). That is why we had one group was PolyA tail treated and the other group was not PolyA tail treated in this experiment.

• This is the information on the barcoded samples:

Barcode03: Leptospira Icterrohaemorrhagica PolyA Tail treated

Barcode04: Leptospira Copenhageni PolyA Tail treated

Barcode05: Leptospira Patoc PolyA Tail treated

Barcode06: Leptospira Mankarso PolyA Tail treated

Barcode07: Leptospira Copenhageni non PolyA Tail treated (using Qiagen RNA Extraction Kit)

Barcode08: Leptospira Icterrohaemorrhagica non PolyA Tail treated

Barcode09: Leptospira Copenhageni non PolyA Tail treated

Barcode10: Leptospira Patoc non PolyA Tail treated

Barcode11: Leptospira Mankarso non PolyA Tail treated

(data info provided by Prakoso,Dhani)

• [x] QC new data
• [x] map new data
• [ ] summary mapping table for new data

[rx32940]

• [x] QC new data
• [x] map new data
• [ ] summary mapping table for new data

• QC results dir:

  /scratch/rx32940/minion_blood_simulation/runs_polyA/QC

• Mapping results: only Lepto, polyA tailed vs non polyA tailed to Lepto:

  
  /scratch/rx32940/minion_blood_simulation/runs_polyA/map/map_lepto

[rx32940]

replicate Alejandro's results on the first trial sequencing data:

• [x] quality check

• [x] map to Lepto reference genome (minimap2)

  • Leptospira_interrogans_Copenhageni_CI & CII (AE016823 & AE016824)
• [ ] map to host reference genome

changed the reference genome to: (https://bacteria.ensembl.org/info/website/ftp/index.html) Leptospira interrogans serovar copenhageni str fiocruz l1 130.ASM768v1.dna.chromosome.I.fa ftp://ftp.ensemblgenomes.org/pub/release-44/bacteria//fasta/bacteria_23_collection/leptospira_interrogans_serovar_copenhageni_str_fiocruz_l1_130/dna/

• also chromosome II reference genome and GTF annotation from this link