Daily Bits - August 2023 – Sam’s Notebook

20230830

Finished CEABIGR gene counts and relatedness matrix stuff.
ProCard transaction verification.

20230829

Worked heavily on CEABIGR gene counts and relatedness matrix.
Worked on lab safety inspection deficiencies.

20230828

Worked on lab safety inspection deficiencies.

20230825

Coral E5 Meeting
Science hour
Updated CEABIGR data using updated Stringtie/Ballgown analysis.

20230824

Pub-a-thon
Lab meeting
Updated CEABIGR data using updated Stringtie/Ballgown analysis.

20230823

In lab:
- Discovered issue with Bioanalyzer priming station.
- Submitted mercuric chloride waste pickup request to EH&S for all 250mL bottles in FTR 228.

20230822

Analysis of initial C.gigas diploids qPCR data for Matt George’s poly:IC experiment

20230821

CEABIGR meeting
- Generate gene/transcript matrices.
- Use matrices to compute distance matrix between samples.
Had constant computer issues most of the day (primarily, VMWare Workstation locking up, due to high CPU usage).
Discovered potential incompatibility between StringTie versions, possibly affecting some downstream operations (namely, running the StringTie prepDE.py3 script), so re-ran the CEABIGR Crassostrea virginica (Eastern oyster) StringTie analysis using the current version of StringTie.

20230818

Caught up on notebook entries
Science Hour

20230817

In lab
- qPCRs of C.gigas diploid poly:IC MgCl₂ control
Helped Shelly get into Raven (GitHub Issue)

sRNA analysis

Concatenate all R1 FastAs

cat /home/shared/8TB_HDD_01/sam/data/A_pulchra/sRNAseq/collapsed/sRNA-ACR-140-145-150-173-178-S1-TP2.flexbar_trim.20230621_1.fastq-collapsed.fasta \
/home/shared/8TB_HDD_01/sam/data/P_evermanni/sRNAseq/collapsed/sRNA-POR-73-79-82-S1-TP2.flexbar_trim.20230621_1.fastq-collapsed.fasta \
/home/shared/8TB_HDD_01/sam/data/P_meandrina/sRNAseq/collapsed/sRNA-POC-47-48-50-53-57-S1-TP2.flexbar_trim.20230621_1.fastq-collapsed.fasta \
>> sRNA-apul-peve-pmea-flexbar_trim.20230621_1-concatenated.fasta

Collapse concatenated FastA to keep only unique sequences:

/home/shared/fastx_toolkit_0.0.13_binaries_Linux_2.6_amd64/bin/fastx_collapser \
-Q 30 \
-i sRNA-apul-peve-pmea-flexbar_trim.20230621_1-concatenated.fasta \
-o sRNA-apul-peve-pmea-flexbar_trim.20230621_1-concatenated.collapsed.fasta

20230816

In lab
- C.gigas Poly:IC
  - Reverse transcribed diploid MgCl₂ control RNA to match previous diploid injected samples.
- Set up Synology RX1217 expansion for Gannet.

20230815

E5 coral sRNA-seq analyses

Collapse trimmed R1 FastQs to non-redundant reads with fastx_collapser to use for cross-wise BLAST-ing:

for fastq in *flexbar*_1.fastq.gz \
do
  filename=${fastq%.*}
  zcat ${fastq} \
  | /home/shared/fastx_toolkit_0.0.13_binaries_Linux_2.6_amd64/bin/fastx_collapser \
  -Q30 \
  -o collapsed/${filename}-collapsed.fasta
done

20230809

P.evermanni sRNA analyses (GitHub Issue)

NCBI BLASTn against miRBase and MirGeneDB, mature and all species, respectively.

Use Perl scripts in miRDeep-P2-pipeline (GitHub repo) to convert/condense FastAs to unique sequnces and T instead of U.
Make NCBI BLAST databases from each.
Concatenate sRNA-seq FastAs (from miRTrace - GitHub Repo) for each sample.

Run NCBI BLASTn

for fasta in /home/shared/8TB_HDD_01/sam/data/P_evermanni/sRNAseq/mirtrace.config.output/qc_passed_reads.all.collapsed/*cat.fasta
do
  filename=${fasta##*/} \
  /home/shared/ncbi-blast-2.11.0+/bin/blastn \
  -db /home/shared/8TB_HDD_01/sam/data/blastdbs/mature_wo_U_uniq.fa \
  -query ${fasta} \
  -out "20230809-peve-miRBase-BLASTn-${filename}.outfmt6" \
  -max_hsps 1 \
  -max_target_seqs 1 \
  -outfmt 6 \
  -num_threads 40
done

for fasta in /home/shared/8TB_HDD_01/sam/data/P_evermanni/sRNAseq/mirtrace.config.output/qc_passed_reads.all.collapsed/*cat.fasta
do 
  filename=${fasta##*/} \
  /home/shared/ncbi-blast-2.11.0+/bin/blastn \
  -db /home/shared/8TB_HDD_01/sam/data/blastdbs/ALL_wo_U_uniq.fas \
  -query ${fasta} \
  -out "20230809-peve-miRGene-BLASTn-${filename}.outfmt6" \
  -max_hsps 1 \
  -max_target_seqs 1 \
  -outfmt 6 \
  -num_threads 40
done

NO MATCHES FOR EITHER!

Run mirdeep2 (GitHub repo- > “About Discovering known and novel miRNAs from small RNA sequencing data”)

First, map reads to genome (*.arf)

for fasta in /home/shared/8TB_HDD_01/sam/data/P_evermanni/sRNAseq/mirtrace.config.output/qc_passed_reads.all.collapsed/no_spaces*.fasta\
  do
  filename=${fasta##*/} \
  /home/shared/mirdeep2/bin/mapper.pl ${fasta} \
  -c \
  -p /home/shared/8TB_HDD_01/sam/data/P_evermanni/genomes/Porites_evermanni_v1 \
  -t "20230809-peve-miRNA-mirdeep2-${filename}.arf"
done

Reformat miRTrace collapsed FastAs to have compatible description lines

for fasta in sRNA*20230621_[12].fasta; do sed '/^>/ s/ /_/g' ${fasta} | sed 's/_rnatype.*$//g' > "no_spaces-${fasta}"; done

Run mirdeep2 on sequencing reads (was having problems with loop, so ran individually)

/home/shared/mirdeep2/bin/miRDeep2.pl \
/home/shared/8TB_HDD_01/sam/data/P_evermanni/sRNAseq/mirtrace.config.output/qc_passed_reads.all.collapsed/no_spaces-sRNA-POR-73-S1-TP2.flexbar_trim.20230621_1.fasta \
/home/shared/8TB_HDD_01/sam/data/P_evermanni/genomes/Porites_evermanni_v1.fa \
20230809-peve-miRNA-mirdeep2-no_spaces-sRNA-POR-73-S1-TP2.flexbar_trim.20230621_1.fasta.arf \
none none none \
2>20230809-peve-miRNA-mirdeep2-no_spaces-sRNA-POR-73-S1-TP2.flexbar_trim.20230621_1.fasta.log

/home/shared/mirdeep2/bin/miRDeep2.pl \
/home/shared/8TB_HDD_01/sam/data/P_evermanni/sRNAseq/mirtrace.config.output/qc_passed_reads.all.collapsed/no_spaces-sRNA-POR-79-S1-TP2.flexbar_trim.20230621_1.fasta \
/home/shared/8TB_HDD_01/sam/data/P_evermanni/genomes/Porites_evermanni_v1.fa \
20230809-peve-miRNA-mirdeep2-no_spaces-sRNA-POR-79-S1-TP2.flexbar_trim.20230621_1.fasta.arf \
none none none \
2>20230809-peve-miRNA-mirdeep2-no_spaces-sRNA-POR-79-S1-TP2.flexbar_trim.20230621_1.fasta.log

/home/shared/mirdeep2/bin/miRDeep2.pl \
/home/shared/8TB_HDD_01/sam/data/P_evermanni/sRNAseq/mirtrace.config.output/qc_passed_reads.all.collapsed/no_spaces-sRNA-POR-82-S1-TP2.flexbar_trim.20230621_1.fasta \
/home/shared/8TB_HDD_01/sam/data/P_evermanni/genomes/Porites_evermanni_v1.fa \
20230809-peve-miRNA-mirdeep2-no_spaces-sRNA-POR-82-S1-TP2.flexbar_trim.20230621_1.fasta.arf \
none none none \
2>20230809-peve-miRNA-mirdeep2-no_spaces-sRNA-POR-82-S1-TP2.flexbar_trim.20230621_1.fasta.log

20230808

P.evermanni sRNA analyses (GitHub Issue)
- MirMachine:
```
MirMachine.py \
--node Metazoa \
--species 20230808-peve-MirMachine \
--genome /home/shared/8TB_HDD_01/sam/data/P_evermanni/genomes/Porites_evermanni_v1.fa \
--cpu 40 \
--add-all-nodes
```
  - 83 loci identified (grep -c "^[^#]" 20230808-peve-MirMachine.PRE.gff)
  - 15 unique miRNA families (grep "^[^#]" 20230808-peve-MirMachine.PRE.gff | awk -F"[\t=;]" '{print $10}'| sort -u | wc -l)

20230804

In lab.
- Fresh install of Ubuntu on swoose. Ugh… Replaced internal HDD (1TB) with 8TB HDD.
- Cleaning for lab safety inspection.
- Lab safety inspection.
- Tried troubleshooting dual boot situation for Jackie’s Linux/Windows computer. Some progress (GRUB menu now shows again), but booting into Windows just hangs at a black screen.

20230803

CEABIGR
- Worked on transcript annotation by exploring differences in DIAMOND/NCBI BLASTx.
- Gathered some prelinary sRNA-seq stats for Steven’s grant update.

20230801