Sam’s Notebook: Transcriptome Assembly – Hematodinium with MEGAN6 Taxonomy-specific Reads with Trinity on Mox

Ran a de novo assembly using [the extracted reads classified under Alveolata from 20200122].(https://ift.tt/2RhkjuM) The assembly was performed with Trinity on Mox.

SBATCH script (GitHub):

#!/bin/bash ## Job Name #SBATCH --job-name=trinity_hemat ## Allocation Definition #SBATCH --account=srlab #SBATCH --partition=srlab ## Resources ## Nodes #SBATCH --nodes=1 ## Walltime (days-hours:minutes:seconds format) #SBATCH --time=10-00:00:00 ## Memory per node #SBATCH --mem=120G ##turn on e-mail notification #SBATCH --mail-type=ALL #SBATCH --mail-user=samwhite@uw.edu ## Specify the working directory for this job #SBATCH --chdir=/gscratch/scrubbed/samwhite/outputs/20200122_hemat_trinity_megan_RNAseq # Exit script if a command fails set -e # Load Python Mox module for Python module availability module load intel-python3_2017 # Document programs in PATH (primarily for program version ID) { date echo "" echo "System PATH for $SLURM_JOB_ID" echo "" printf "%0.s-" {1..10} echo "${PATH}" | tr : \\n } >> system_path.log # User-defined variables reads_dir=/gscratch/srlab/sam/data/C_bairdi/RNAseq threads=27 assembly_stats=assembly_stats.txt timestamp=$(date +%Y%m%d) fasta_name="${timestamp}.hemat.megan.Trinity.fasta" # Paths to programs trinity_dir="/gscratch/srlab/programs/trinityrnaseq-v2.9.0" samtools="/gscratch/srlab/programs/samtools-1.10/samtools" ## Inititalize arrays R1_array=() R2_array=() # Variables for R1/R2 lists R1_list="" R2_list="" # Create array of fastq R1 files R1_array=(${reads_dir}/*_R1.fq) # Create array of fastq R2 files R2_array=(${reads_dir}/*_R2.fq) # Create list of fastq files used in analysis ## Uses parameter substitution to strip leading path from filename for fastq in ${reads_dir}/*.fq do echo "${fastq##*/}" >> fastq.list.txt done # Create comma-separated lists of FastQ reads R1_list=$(echo "${R1_array[@]}" | tr " " ",") R2_list=$(echo "${R2_array[@]}" | tr " " ",") # Run Trinity using "stranded" setting (--SS_lib_type) ${trinity_dir}/Trinity \ --seqType fq \ --max_memory 120G \ --CPU ${threads} \ --SS_lib_type RF \ --left "${R1_list}" \ --right "${R2_list}" # Rename generic assembly FastA mv trinity_out_dir/Trinity.fasta trinity_out_dir/${fasta_name} # Assembly stats ${trinity_dir}/util/TrinityStats.pl trinity_out_dir/${fasta_name} \ > ${assembly_stats} # Create gene map files ${trinity_dir}/util/support_scripts/get_Trinity_gene_to_trans_map.pl \ trinity_out_dir/${fasta_name} \ > trinity_out_dir/${fasta_name}.gene_trans_map # Create FastA index ${samtools} faidx \ trinity_out_dir/${fasta_name} 

Sam’s Notebook: Transcriptome Assembly – C.bairdi with MEGAN6 Taxonomy-specific Reads with Trinity on Mox

Ran a de novo assembly using [the extracted reads classified under Arthropoda from 20200122].(https://ift.tt/2RhkjuM) The assembly was performed with Trinity on Mox.

SBATCH Script (GitHub):

#!/bin/bash ## Job Name #SBATCH --job-name=trinity_cbai ## Allocation Definition #SBATCH --account=srlab #SBATCH --partition=srlab ## Resources ## Nodes #SBATCH --nodes=1 ## Walltime (days-hours:minutes:seconds format) #SBATCH --time=10-00:00:00 ## Memory per node #SBATCH --mem=500G ##turn on e-mail notification #SBATCH --mail-type=ALL #SBATCH --mail-user=samwhite@uw.edu ## Specify the working directory for this job #SBATCH --chdir=/gscratch/scrubbed/samwhite/outputs/20200122_cbai_trinity_megan_RNAseq # Exit script if a command fails set -e # Load Python Mox module for Python module availability module load intel-python3_2017 # Document programs in PATH (primarily for program version ID) { date echo "" echo "System PATH for $SLURM_JOB_ID" echo "" printf "%0.s-" {1..10} echo "${PATH}" | tr : \\n } >> system_path.log # User-defined variables reads_dir=/gscratch/srlab/sam/data/C_bairdi/RNAseq threads=27 assembly_stats=assembly_stats.txt timestamp=$(date +%Y%m%d) fasta_name="${timestamp}.C_bairdi.megan.Trinity.fasta" # Paths to programs trinity_dir="/gscratch/srlab/programs/trinityrnaseq-v2.9.0" samtools="/gscratch/srlab/programs/samtools-1.10/samtools" ## Inititalize arrays R1_array=() R2_array=() # Variables for R1/R2 lists R1_list="" R2_list="" # Create array of fastq R1 files R1_array=(${reads_dir}/*_R1.fq) # Create array of fastq R2 files R2_array=(${reads_dir}/*_R2.fq) # Create list of fastq files used in analysis ## Uses parameter substitution to strip leading path from filename for fastq in ${reads_dir}/*.fq do echo "${fastq##*/}" >> fastq.list.txt done # Create comma-separated lists of FastQ reads R1_list=$(echo "${R1_array[@]}" | tr " " ",") R2_list=$(echo "${R2_array[@]}" | tr " " ",") # Run Trinity using "stranded" setting (--SS_lib_type) ${trinity_dir}/Trinity \ --seqType fq \ --max_memory 500G \ --CPU ${threads} \ --SS_lib_type RF \ --left "${R1_list}" \ --right "${R2_list}" # Rename generic assembly FastA mv trinity_out_dir/Trinity.fasta trinity_out_dir/${fasta_name} # Assembly stats ${trinity_dir}/util/TrinityStats.pl trinity_out_dir/${fasta_name} \ > ${assembly_stats} # Create gene map files ${trinity_dir}/util/support_scripts/get_Trinity_gene_to_trans_map.pl \ trinity_out_dir/${fasta_name} \ > trinity_out_dir/${fasta_name}.gene_trans_map # Create FastA index ${samtools} faidx \ trinity_out_dir/${fasta_name} 

Sam’s Notebook: RNA Isolation and Quantification – C.bairdi Hemocyte Pellets in RNAlater Troubleshooting

After the failure to obtain RNA from any C.bairdi hemocytes pellets (out of 24 samples processed) on 20200117, I decided to isolate RNA from just a subset of that group to determine if I screwed something up last time or something. Also, I am testing two different preparations of the kit-supplied DNase I: one Kaitlyn prepped and a fresh preparation that I made. Admittedly, I’m not doing the “proper” testing by trying the different DNase preps on the same exact sample, but it’ll do. I just want to see if I get some RNA from these samples this time…

Isolated RNA from the following 4 hemolymph pellet samples:

  • 6128_112_9 (Kaitlyn DNase)
  • 6204_114_9 (Kaitlyn DNase)
  • 6141_123_9 (Sam DNase)
  • 6245_126_9 (Sam DNase)

Isolated RNA using the Quick DNA/RNA Microprep Kit (ZymoResearch; PDF) according to the manufacturer’s protocol for liquids/cells in RNAlater.

  • Used 35uL from each RNAlater/hemocyte slurry.
  • Mixed with equal volume of H2O (35uL).
  • Retained DNA on the Zymo-Spin IC-XM columns for isolation after RNA isolation.
  • Performed on-column DNase step.
  • RNA was eluted in 15uL H2O

RNA was quantified on the Roberts Lab Qubit 3.0 using the RNA High Sensitivity Assay (Invitrogen), using 2uL of each sample.

Kaitlyn’s notebook: Geoduck reproductive development primer design

After testing the previous primers, it was decided that the primers were not specific enough for successful sex or reproductive stage identification. I am going to design new primers with the most recent geoduck genome version of all proteins listed in Fig. 6.

I am following Sam’s Jupyter post  (downloadable here) and Github post. I am focusing on primer design and primer specificity, as the fasta file containing the 8 proteins in Fig 6. has been created.

I used jupyter notebook to annotate code and explain shell to understand new commands.

        1. Download
          • pyfaidx – splits multi-FastA into individual FastA files
          • Primer3 – performs primer design
          • EMBOSS(installation issue linked) – checks primer specificity against entire transcriptome
            • installed without X11 support (problem with X11 headers)
        2. Split FastA into multiple FastAs using pyfaidx
        3. Design primers with Primer3 (issue with code)
        4. Check specificity using EMBOSS
          • Problem with primersearch function

 

Sam’s Notebook: DNA Quality Assessment – Agarose Gel for C.bairdi 2010255-2729 gDNA from 20200122

Earlier today, I isolated gDNA from C.bairdi 20102558-2729 ethanol-preserved muscle tissue using the Quick DNA/RNA MicroPrep Plus Kit (ZymoResearch) and prepared the tissue in three different ways to see how they would compare:

Ran 10uL of each sample on a 0.8% agarose, 1x low TAE gel with ethidium bromide.

Used the GeneRuler DNA Ladder Mix (ThermoFisher) as ladder.

Sam’s Notebook: DNA Isolation – C.bairdi 20102558-2729 EtOH-preserved Tissue via Three Variations Using Quick DNA-RNA MicroPrep Kit

Previously, I isolated gDNA from a C.bairdi EtOH-preserved muscle sample (20102558-2729) on 20200108 using the E.Z.N.A. Mollusc DNA Kit (Omega). Although the yields were excellent, the DNA looked completely degraded on a gel and running that DNA on a minION flowcell yielded relatively short reads (which wasn’t terribly surprising).

So, to test out whether or not the degradation was due to the inherent age/preservation method of the sample, I decided to try another kit for gDNA isolation: Quick DNA/RNA MicroPrep Kit (ZymoResearch). I also decided to try three variations of the isolation protocol with very small pieces of tissue (~5 – 10mg):

  • “damp”: where the ethanol has not been allowed to dry and homogenized with disposable mortar/pestle 1.5mL tube in 800uL of 1x DNA/RNA Shield.
  • “dry”: where the sample is allowed to dry (per the “FFPE Tissue” guidelines) and homogenized with disposable mortar/pestle 1.5mL tube in 800uL of 1x DNA/RNA Shield.
  • minced: where the damp sample was minced with a razor blade and incubate in 300uL of 1x DNA/RNA Shield with 30uL PK Digestion Buffer and 15uL of Proteinase K solution at 55oC for 5hrs.

The remainder of the manufacturer’s protocol was followed.

Samples were eluted in 30uL of H2O.

Samples were quantified on the Roberts Lab Qubit 3.0 using 1uL of each sample with the 1x dsDNA High Sensitivity Assay.

Shelly’s Notebook: Wed. Jan. 22, 2020 KCl in Geoduck Strip Spawn

Does priming with KCl matter?

This was a small scale experiment performed to determine if fertilization can be achieved without KCl priming.

Experimental setup:

  • ~10mL of eggs prior to KCl priming
  • ~10mL of eggs prior to KCl priming + 3 drops of sperm (fertilization started at 2:35pm)
  • ~300uL of KCl primed eggs + sperm mix (600mL eggs + 6mL sperm) (fertilization started at 2:54pm)

RESULTS:

  • No Sperm, No KCl (90 min post collection): uc?export=view&id=11rXlDFYUJ0Wtmi4hdgStHdJtd0qPmK2J

uc?export=view&id=12OUDupaEHcN9OuWdPVnqkfFKwLI3dN3b

  • +Sperm, No KCl (90 min post collection, 70 min post-fertilization):
    uc?export=view&id=1wh8BwgPzqT5lXcmdh_YG6Uh_LWgIkhkI

uc?export=view&id=12Zm08ok7MktpYt7RwkQagk5iQtCa8rjg

  • +Sperm, +KCl (60 min post-fertilization):

uc?export=view&id=1_NIXDvOGT-MeqwgIgHY03thtMkdHUNvR

uc?export=view&id=1gWtmM42G76g2C5T12ZzOhEsuEQmzNtnw

Incubated samples over night at 4C

  • No Sperm, No KCl (~21 hours post collection, 100uL drop on slide):
    uc?export=view&id=1NVuM1TRBt3NN8YXC6ddKKxKosJcu8_M2

uc?export=view&id=1NrT0bzhlIQXBZofDyO9F0eScfYSm0OaP

uc?export=view&id=1CiEKIwxm9E2rv8e8UXKXDkxbmwUeGXcb

uc?export=view&id=1LP0C_a7X1dbe0jEeufk9biBtnaa4NdZM

  • +Sperm, No KCl (~21 hours post collection, ~20.5 hours post fertilization, 100uL drop on slide):

uc?export=view&id=19VLvpMA9o6D6fTGYC8-Cr2c9O0NsjpUC

uc?export=view&id=1o71ve4tvb0dZZxFgvdz0rU8dDGWiXtsQ

uc?export=view&id=1C1SH-6qrEw7VXt3_V-YxRkHUr7uXAmiX

uc?export=view&id=1vdHHewW5LydPU_87jAUVqUvVGrrUMqnW

uc?export=view&id=1SFHEYbozigD-ONkya_bniPx8RdxcfHFZ

  • +Sperm, +KCl (~20.5 hours post fertilization, ~50uL drop on slide because that’s all that was left):

uc?export=view&id=18a5RacUvtiv_WveiQ1XYehuSrwmjOK9k uc?export=view&id=1RghO_psdRXQZCjRDNelt66LPT4zHltwm uc?export=view&id=1PQ878QMLDAj8jfHJW6Rz3Ui8H5WhXPp0 uc?export=view&id=1pegQe1O7SkKLD4RwOTaH5gjcC2N6td1C uc?export=view&id=1T5ztjoTNI409Z8jJAkazgvVnRk5BpuyI uc?export=view&id=1sw5qoO6m3mpOoZUdyNYe8YoRAuXjcHsj uc?export=view&id=1fp3je2-zsL-355HJQPfrps5rOo61YGsr

Link to all 20200121 photos

Link to all 20200122 photos

CONCLUSIONS

  • It seems like no embryo development happened in the no Sperm, no KCl sample
  • It seems like some embryo development did happen in the Sperm, no KCl sample after letting the fertilization go overnight, but they look kind of weird. I’m not sure if algae is eating them?
  • It seems like some embryo development did happen in the Sperm, no KCl sample after letting the fertilization go overnight. Most of these eggs seemed to lose pigment (does that mean they are dead or dying?)

Will have to discuss results with group

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