Daily Bits - August 2023

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