Shelly’s Notebook: Wed. Jul. 10, 2019 Salmon-Sea lice Zymo Pico Methyl prep + RRBS digest repeat

Zymo Pico Methyl kit prep

  • set up programs on PCR machine in 209 (called Zym1-4 under STRIGG folder)
  • Into a 48-well plate, I aliquoted out 20-50ng DNA and up to 20uL of nanopure H2O according to this sheet
  • I added 50ng of sea lice DNA:
    • Sea lice Female 1 = 67.2ng/uL (qubit HS, 7/10)
      • added 0.75uL + 19.25uL nanopure H2O
    • Sea lice Female 2 = 17ng/ul (qubit HS, 7/10)
      • added 3uL + 17uL nanopure H2O

Following Zymo pico methyl kit protocol section 1:

  • Added 130uL of lightening conversion reagent to all wells and incubated at 98C 8 min, 54C 1 hour, hold @4C
  • I combined all samples + 600uL of ***DNA binding buffer into Zymo DNA concentrator columns, spun, discarded supernatent
  • added 200uL desulphonation buffer to all columns and incubated at RT 20 min.

*** I realized during the incubation that instead of DNA binding buffer, this was suppose to be M-binding buffer!!! So I called Zymo tech support and was advised to not proceed with the kit because the yield could be poor in quantity and size and Zymo couldn’t guarentee the libraries would be representative. DNA binding buffer is not optimized for single stranded DNA (which after the conversion reagent, the DNA is). The DNA binding buffer can be used at a 6-7:1 ratio with ssDNA but that I not what I did. If I had kept the flow through I could have attempted to re-bind it to the column with the m-binding buffer.

SO, I took Zymo’s advice, and ordered more lightening conversion reagent to attempt the preps tomorrow. But I needed to repeat the digests to have enough DNA to start the preps with. BUMMER! But better that than get crappy data.

Repeating RRBS

  • Followed digest and size selection plan exactly as outlined here with the following exceptions:
    • I prepared reactions in a 48-well PCR plate
      • I first prepared the MSPI master mix and added it to all wells on ice. Then added DNA and water if needed
    • I created a program on the PCR machine in 209 for the incubations (called “RRBS” under the folder STRIGG).
    • MSPI digest incubated @37C for 1 hour
    • I paused the program to add 10uL of TAQ-a1 master mix/well
    • Then resumed the program to incubate at 65C for 30 min.
  • I measured DNA concentrations (S2 read 9.98ng/uL at the beginning and then 9.88 at the end)
    • DNA concentrations are here
  • I aliquoted 22-50ng salmon DNA into a 48-well plate following the volumes listed here and froze the plate at -20C

####plan for tomorrow

  • aliquot sea lice DNA into the plate
  • attempt all of Zymo pico methyl kit prep

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Shelly’s Notebook: Tues. Jul. 9, 2019 Salmon RRBS + Zymo Pico Methyl prep

RRBS

  • Followed digest and size selection plan exactly as outlined here
  • DNA yield after digestion and size selection ranged from 20-110ng, which is on par with what I got in the June 28 test run
    • concentration and yields can be found here
    • Digested size selceted samples (tubes labelled “sz gDNA 1-20”) are stored in 209 -20C bottom drawer in box labeled “gDNA Salmon Skin sea lice infection (from Christian) S.Trigg”

Zymo Pico Methyl kit prep

  • picked up primers today from Biochem stores
  • reconstituted in EB (Qiagen) @ 100uM
  • added them to the primer stocks inventory
  • prepared 50uL of 10uM Index primer mixes following Karolyn’s instructions:
    • 50uL Index Primer 10uM (section 5) =
      • 5uL 100uM Ill_TrSq_P5_Univ
      • 5uL 100uM Ill_TrSq_P7_# (where # = index 1-22)
      • 40uL EB
    • We now have Index Primer mixes 1-22 with #2,4,5,6,7,and 12 coming from the kit and all others coming from IDT/I made.

Plan for tomorrow

  • Set up programs on PCR machine
  • Aliquot out up to 50ng Salmon DNA (see volumes here)
  • Aliquot out 50ng of sea lice DNA
  • start library prep

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Sam’s Notebook: Genome Assessment – BUSCO Metazoa on Pgenerosa_v074 on Mox

Ran BUSCO on Mox for our Pgenerosa_v74 genome assembly to assess “completeness”. This is the assembly that only has the longest 18 scaffolds (the scaffolds hand-curated by Phase Genomics).

SBATCH script (GitHub):

 #!/bin/bash ## Job Name #SBATCH --job-name=busco_pgen74 ## Allocation Definition #SBATCH --account=srlab #SBATCH --partition=srlab ## Resources ## Nodes #SBATCH --nodes=1 ## Walltime (days-hours:minutes:seconds format) #SBATCH --time=15-00:00:00 ## 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 --workdir=/gscratch/scrubbed/samwhite/outputs/20190710_busco_pgen_v074_unannotated # Load Python Mox module for Python module availability module load intel-python3_2017 # Load Open MPI module for parallel, multi-node processing module load icc_19-ompi_3.1.2 # SegFault fix? export THREADS_DAEMON_MODEL=1 # Document programs in PATH (primarily for program version ID) date >> system_path.log echo "" >> system_path.log echo "System PATH for $SLURM_JOB_ID" >> system_path.log echo "" >> system_path.log printf "%0.s-" {1..10} >> system_path.log echo "${PATH}" | tr : \\n >> system_path.log # Establish variables for more readable code ## Input files and settings base_name=Pgenerosa_v074 busco_db=/gscratch/srlab/sam/data/databases/BUSCO/metazoa_odb9 genome_fasta=/gscratch/srlab/sam/data/P_generosa/genomes/Pgenerosa_v074.fa augustus_species=fly threads=28 ## Save working directory wd=$(pwd) ## Set program paths augustus_bin=/gscratch/srlab/programs/Augustus-3.3.2/bin augustus_scripts=/gscratch/srlab/programs/Augustus-3.3.2/scripts blast_dir=/gscratch/srlab/programs/ncbi-blast-2.8.1+/bin/ busco=/gscratch/srlab/programs/busco-v3/scripts/run_BUSCO.py hmm_dir=/gscratch/srlab/programs/hmmer-3.2.1/src/ ## Augustus configs augustus_dir=${wd}/augustus augustus_config_dir=${augustus_dir}/config augustus_orig_config_dir=/gscratch/srlab/programs/Augustus-3.3.2/config ## BUSCO configs busco_config_default=/gscratch/srlab/programs/busco-v3/config/config.ini.default busco_config_ini=${wd}/config.ini # Export BUSCO config file location export BUSCO_CONFIG_FILE="${busco_config_ini}" # Export Augustus variable export PATH="${augustus_bin}:$PATH" export PATH="${augustus_scripts}:$PATH" export AUGUSTUS_CONFIG_PATH="${augustus_config_dir}" # Copy BUSCO config file cp ${busco_config_default} "${busco_config_ini}" # Make Augustus directory if it doesn't exist if [ ! -d "${augustus_dir}" ]; then mkdir --parents "${augustus_dir}" fi # Copy Augustus config directory cp --preserve -r ${augustus_orig_config_dir} "${augustus_dir}" # Edit BUSCO config file ## Set paths to various programs ### The use of the % symbol sets the delimiter sed uses for arguments. ### Normally, the delimiter that most examples use is a slash "/". ### But, we need to expand the variables into a full path with slashes, which screws up sed. ### Thus, the use of % symbol instead (it could be any character that is NOT present in the expanded variable; doesn't have to be "%"). sed -i "/^;cpu/ s/1/${threads}/" "${busco_config_ini}" sed -i "/^tblastn_path/ s%tblastn_path = /usr/bin/%path = ${blast_dir}%" "${busco_config_ini}" sed -i "/^makeblastdb_path/ s%makeblastdb_path = /usr/bin/%path = ${blast_dir}%" "${busco_config_ini}" sed -i "/^augustus_path/ s%augustus_path = /home/osboxes/BUSCOVM/augustus/augustus-3.2.2/bin/%path = ${augustus_bin}%" "${busco_config_ini}" sed -i "/^etraining_path/ s%etraining_path = /home/osboxes/BUSCOVM/augustus/augustus-3.2.2/bin/%path = ${augustus_bin}%" "${busco_config_ini}" sed -i "/^gff2gbSmallDNA_path/ s%gff2gbSmallDNA_path = /home/osboxes/BUSCOVM/augustus/augustus-3.2.2/scripts/%path = ${augustus_scripts}%" "${busco_config_ini}" sed -i "/^new_species_path/ s%new_species_path = /home/osboxes/BUSCOVM/augustus/augustus-3.2.2/scripts/%path = ${augustus_scripts}%" "${busco_config_ini}" sed -i "/^optimize_augustus_path/ s%optimize_augustus_path = /home/osboxes/BUSCOVM/augustus/augustus-3.2.2/scripts/%path = ${augustus_scripts}%" "${busco_config_ini}" sed -i "/^hmmsearch_path/ s%hmmsearch_path = /home/osboxes/BUSCOVM/hmmer/hmmer-3.1b2-linux-intel-ia32/binaries/%path = ${hmm_dir}%" "${busco_config_ini}" # Run BUSCO/Augustus training ${busco} \ --in ${genome_fasta} \ --out ${base_name} \ --lineage_path ${busco_db} \ --mode genome \ --cpu ${threads} \ --long \ --species ${augustus_species} \ --tarzip \ --augustus_parameters='--progress=true'  

Sam’s Notebook: Genome Assessment – BUSCO Metazoa on Pgenerosa_v70 on Mox

Ran BUSCO on Mox for our Pgenerosa_v70 genome assembly to assess “completeness”.

SBATCH script (GitHub):

 #!/bin/bash ## Job Name #SBATCH --job-name=busco_pgen70 ## Allocation Definition #SBATCH --account=srlab #SBATCH --partition=srlab ## Resources ## Nodes #SBATCH --nodes=1 ## Walltime (days-hours:minutes:seconds format) #SBATCH --time=15-00:00:00 ## 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 --workdir=/gscratch/scrubbed/samwhite/outputs/20190710_busco_pgen_v070 # Load Python Mox module for Python module availability module load intel-python3_2017 # Load Open MPI module for parallel, multi-node processing module load icc_19-ompi_3.1.2 # SegFault fix? export THREADS_DAEMON_MODEL=1 # Document programs in PATH (primarily for program version ID) date >> system_path.log echo "" >> system_path.log echo "System PATH for $SLURM_JOB_ID" >> system_path.log echo "" >> system_path.log printf "%0.s-" {1..10} >> system_path.log echo "${PATH}" | tr : \\n >> system_path.log # Establish variables for more readable code ## Input files and settings base_name=Pgenerosa_v070 busco_db=/gscratch/srlab/sam/data/databases/BUSCO/metazoa_odb9 genome_fasta=/gscratch/srlab/sam/data/P_generosa/genomes/Pgenerosa_v070.fa augustus_species=fly threads=28 ## Save working directory wd=$(pwd) ## Set program paths augustus_bin=/gscratch/srlab/programs/Augustus-3.3.2/bin augustus_scripts=/gscratch/srlab/programs/Augustus-3.3.2/scripts blast_dir=/gscratch/srlab/programs/ncbi-blast-2.8.1+/bin/ busco=/gscratch/srlab/programs/busco-v3/scripts/run_BUSCO.py hmm_dir=/gscratch/srlab/programs/hmmer-3.2.1/src/ ## Augustus configs augustus_dir=${wd}/augustus augustus_config_dir=${augustus_dir}/config augustus_orig_config_dir=/gscratch/srlab/programs/Augustus-3.3.2/config ## BUSCO configs busco_config_default=/gscratch/srlab/programs/busco-v3/config/config.ini.default busco_config_ini=${wd}/config.ini # Export BUSCO config file location export BUSCO_CONFIG_FILE="${busco_config_ini}" # Export Augustus variable export PATH="${augustus_bin}:$PATH" export PATH="${augustus_scripts}:$PATH" export AUGUSTUS_CONFIG_PATH="${augustus_config_dir}" # Copy BUSCO config file cp ${busco_config_default} "${busco_config_ini}" # Make Augustus directory if it doesn't exist if [ ! -d "${augustus_dir}" ]; then mkdir --parents "${augustus_dir}" fi # Copy Augustus config directory cp --preserve -r ${augustus_orig_config_dir} "${augustus_dir}" # Edit BUSCO config file ## Set paths to various programs ### The use of the % symbol sets the delimiter sed uses for arguments. ### Normally, the delimiter that most examples use is a slash "/". ### But, we need to expand the variables into a full path with slashes, which screws up sed. ### Thus, the use of % symbol instead (it could be any character that is NOT present in the expanded variable; doesn't have to be "%"). sed -i "/^;cpu/ s/1/${threads}/" "${busco_config_ini}" sed -i "/^tblastn_path/ s%tblastn_path = /usr/bin/%path = ${blast_dir}%" "${busco_config_ini}" sed -i "/^makeblastdb_path/ s%makeblastdb_path = /usr/bin/%path = ${blast_dir}%" "${busco_config_ini}" sed -i "/^augustus_path/ s%augustus_path = /home/osboxes/BUSCOVM/augustus/augustus-3.2.2/bin/%path = ${augustus_bin}%" "${busco_config_ini}" sed -i "/^etraining_path/ s%etraining_path = /home/osboxes/BUSCOVM/augustus/augustus-3.2.2/bin/%path = ${augustus_bin}%" "${busco_config_ini}" sed -i "/^gff2gbSmallDNA_path/ s%gff2gbSmallDNA_path = /home/osboxes/BUSCOVM/augustus/augustus-3.2.2/scripts/%path = ${augustus_scripts}%" "${busco_config_ini}" sed -i "/^new_species_path/ s%new_species_path = /home/osboxes/BUSCOVM/augustus/augustus-3.2.2/scripts/%path = ${augustus_scripts}%" "${busco_config_ini}" sed -i "/^optimize_augustus_path/ s%optimize_augustus_path = /home/osboxes/BUSCOVM/augustus/augustus-3.2.2/scripts/%path = ${augustus_scripts}%" "${busco_config_ini}" sed -i "/^hmmsearch_path/ s%hmmsearch_path = /home/osboxes/BUSCOVM/hmmer/hmmer-3.1b2-linux-intel-ia32/binaries/%path = ${hmm_dir}%" "${busco_config_ini}" # Run BUSCO/Augustus training ${busco} \ --in ${genome_fasta} \ --out ${base_name} \ --lineage_path ${busco_db} \ --mode genome \ --cpu ${threads} \ --long \ --species ${augustus_species} \ --tarzip \ --augustus_parameters='--progress=true'  

Laura’s Notebook: Starting RNA isolation for Oly OA project

Today I began a big round of RNA isolation, which I will eventually use for QuantSeq libraries and sequence for gene expression. RNA will be isolated using RNAzol, from adult Oly ctenidia tissue after 7 weeks in high/ambient pCo2, and from newly released larvae from parents who had previously been exposed to pCO2 treatments. Larvae are pooled by a daily larval pulse.

Step 1. Homogenize tissues.

I’m homogenizing tissues in liquid nitrogen with mortar + pestle. It’s rather labor intensive, and I lose lots of my tissues, however it’s the best way to fully homogenize, particularly the larvae.

Today I began with ctendia tissues.

  • Added 1mL RNAzol to 1.5 mL microcentrifuge tubes.
  • Cleaned mortars, pestles, and metal spatulas. Did this by cleaning under hot water, soaking in 10% bleach/DI solution for a minimum of 10 minutes, rinsing thoroughly with DI water, then rinsing with 190 proof ethanol and letting dry.
  • Put tubes with RNAzol on scale. Ground tissues to powder, scraped with metal spatula and carefully transferred powder to tube. The goal was 100 mg, but my tissues were mostly too small. I typcially achieved ~20-60 mg.

I did 8 samples at a time (the # of mortar+pestle kits I have), then cleaned and repeated with another 8. Each round of 8 included 2 samples from different cohort + pCO2 treatment. I’m doing the Oyster Bay, Fidalgo Bay and Dabob Bay F1 cohorts.

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