Grace’s Notebook: Rna Isolation Third Batch With Qubit Results

RNA Isolation

I had previously thought that RNAzol was a dye that binded to RNA… I double-checked with Sam, because I realized I never really asked, and after centrifuging, the RNAzol (blue) is often at the top of the tube, but sometimes it’s in the middle or towards the bottom and I wasn’t sure if I should be aiming for the blue when removing the supernatant for further processing. He said the RNAzol is actually salts and other stuff that helps RNA become isolated. So, when taking out the supernatant, it’s ok if I get some blue, but I should aim for the clear liquid, while avoiding the bottom, becasue that’s where the junk gets pelleted down to.

Today I ran 9 more samples: img

I had a couple of mishaps today 😦 Firstly, during the step where I remove the supernatant into a new tube, I accidentally put the tip of the pipet too far down, and I think I likely got some junk in with the supernatant for tube # 474. Luckily tube 474 is from the third hemolymph sampling day, which means that I have two more samples from that same crab for that day.

Secondly, when adding 750µL of 2-propanol, I accidentally tipped tube #38 (first sampling day) and lost some of the supernatant that I had saved.

Here is Sam’s notebook that I should have looked at wayyyy before starting this process. I had forgotten that he did this on two crab samples a while back. link

I see that Sam mixed the 2-propanol with the supernatant by inversion rather than vortex. I think I will start doing this instead, because the vortex is so powerful that the propanol often ends up leaking a little, and smearing the tube labels.

Qubit results

Used 5µL of sample from tubes 65 and 498. 65 was out of range, 498 was 1.8ng/µL. img

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Grace’s Notebook: Rna Isolation Third Batch

RNA Isolation

I had previously thought that RNAzol was a dye that binded to RNA… I double-checked with Sam, because I realized I never really asked, and after centrifuging, the RNAzol (blue) is often at the top of the tube, but sometimes it’s in the middle or towards the bottom and I wasn’t sure if I should be aiming for the blue when removing the supernatant for further processing. He said the RNAzol is actually salts and other stuff that helps RNA become isolated. So, when taking out the supernatant, it’s ok if I get some blue, but I should aim for the clear liquid, while avoiding the bottom, becasue that’s where the junk gets pelleted down to.

Today I ran 9 more samples: img

I had a couple of mishaps today 😦 Firstly, during the step where I remove the supernatant into a new tube, I accidentally put the tip of the pipet too far down, and I think I likely got some junk in with the supernatant for tube # 474. Luckily tube 474 is from the third hemolymph sampling day, which means that I have two more samples from that same crab for that day.

Secondly, when adding 750µL of 2-propanol, I accidentally tipped tube #38 (first sampling day) and lost some of the supernatant that I had saved.

Here is Sam’s notebook that I should have looked at wayyyy before starting this process. I had forgotten that he did this on two crab samples a while back. link

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Sam’s Notebook:Assembly – Geoduck NovaSeq using SparseAssembler kmer = 101

0000-0002-2747-368X

The prior run used a kmer size of 61, and the resulting assembly was rather poor (small N50).

For this run, I arbitrarily increased the kmer size to 101, in hopes that this will improve the assembly.

The job was run on our Mox node.

Here’s the batch script to initiate the job:

20180322_SparseAssembler_novaseq_geoduck_slurm.sh

 #!/bin/bash ## Job Name #SBATCH --job-name=20180322_sparse_assembler_geo_novaseq ## Allocation Definition #SBATCH --account=srlab #SBATCH --partition=srlab ## Resources ## Nodes (We only get 1, so this is fixed) #SBATCH --nodes=1 ## Walltime (days-hours:minutes:seconds format) #SBATCH --time=30-00:00:00 ## 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 --workdir=/gscratch/scrubbed/samwhite/20180322_SparseAssembler_novaseq_geoduck /gscratch/srlab/programs/SparseAssembler/SparseAssembler LD 0 NodeCovTh 1 EdgeCovTh 0 k 101 g 15 PathCovTh 100 GS 2200000000 i1 /gscratch/scrubbed/samwhite/20180129_trimmed_again/AD002_S9_L001_R1_001_val_1_val_1.fastq i2 /gscratch/scrubbed/samwhite/20180129_trimmed_again/AD002_S9_L001_R2_001_val_2_val_2.fastq i1 /gscratch/scrubbed/samwhite/20180129_trimmed_again/AD002_S9_L002_R1_001_val_1_val_1.fastq i2 /gscratch/scrubbed/samwhite/20180129_trimmed_again/AD002_S9_L002_R2_001_val_2_val_2.fastq i1 /gscratch/scrubbed/samwhite/20180129_trimmed_again/NR005_S4_L001_R1_001_val_1_val_1.fastq i2 /gscratch/scrubbed/samwhite/20180129_trimmed_again/NR005_S4_L001_R2_001_val_2_val_2.fastq i1 /gscratch/scrubbed/samwhite/20180129_trimmed_again/NR005_S4_L002_R1_001_val_1_val_1.fastq i2 /gscratch/scrubbed/samwhite/20180129_trimmed_again/NR005_S4_L002_R2_001_val_2_val_2.fastq i1 /gscratch/scrubbed/samwhite/20180129_trimmed_again/NR006_S3_L001_R1_001_val_1_val_1.fastq i2 /gscratch/scrubbed/samwhite/20180129_trimmed_again/NR006_S3_L001_R2_001_val_2_val_2.fastq i1 /gscratch/scrubbed/samwhite/20180129_trimmed_again/NR006_S3_L002_R1_001_val_1_val_1.fastq i2 /gscratch/scrubbed/samwhite/20180129_trimmed_again/NR006_S3_L002_R2_001_val_2_val_2.fastq i1 /gscratch/scrubbed/samwhite/20180129_trimmed_again/NR012_S1_L001_R1_001_val_1_val_1.fastq i2 /gscratch/scrubbed/samwhite/20180129_trimmed_again/NR012_S1_L001_R2_001_val_2_val_2.fastq i1 /gscratch/scrubbed/samwhite/20180129_trimmed_again/NR012_S1_L002_R1_001_val_1_val_1.fastq i2 /gscratch/scrubbed/samwhite/20180129_trimmed_again/NR012_S1_L002_R2_001_val_2_val_2.fastq i1 /gscratch/scrubbed/samwhite/20180129_trimmed_again/NR013_AD013_S2_L001_R1_001_val_1_val_1.fastq i2 /gscratch/scrubbed/samwhite/20180129_trimmed_again/NR013_AD013_S2_L001_R2_001_val_2_val_2.fastq i1 /gscratch/scrubbed/samwhite/20180129_trimmed_again/NR013_AD013_S2_L002_R1_001_val_1_val_1.fastq i2 /gscratch/scrubbed/samwhite/20180129_trimmed_again/NR013_AD013_S2_L002_R2_001_val_2_val_2.fastq i1 /gscratch/scrubbed/samwhite/20180129_trimmed_again/NR014_AD014_S5_L001_R1_001_val_1_val_1.fastq i2 /gscratch/scrubbed/samwhite/20180129_trimmed_again/NR014_AD014_S5_L001_R2_001_val_2_val_2.fastq i1 /gscratch/scrubbed/samwhite/20180129_trimmed_again/NR014_AD014_S5_L002_R1_001_val_1_val_1.fastq i2 /gscratch/scrubbed/samwhite/20180129_trimmed_again/NR014_AD014_S5_L002_R2_001_val_2_val_2.fastq i1 /gscratch/scrubbed/samwhite/20180129_trimmed_again/NR015_AD015_S6_L001_R1_001_val_1_val_1.fastq i2 /gscratch/scrubbed/samwhite/20180129_trimmed_again/NR015_AD015_S6_L001_R2_001_val_2_val_2.fastq i1 /gscratch/scrubbed/samwhite/20180129_trimmed_again/NR015_AD015_S6_L002_R1_001_val_1_val_1.fastq i2 /gscratch/scrubbed/samwhite/20180129_trimmed_again/NR015_AD015_S6_L002_R2_001_val_2_val_2.fastq i1 /gscratch/scrubbed/samwhite/20180129_trimmed_again/NR019_S7_L001_R1_001_val_1_val_1.fastq i2 /gscratch/scrubbed/samwhite/20180129_trimmed_again/NR019_S7_L001_R2_001_val_2_val_2.fastq i1 /gscratch/scrubbed/samwhite/20180129_trimmed_again/NR019_S7_L002_R1_001_val_1_val_1.fastq i2 /gscratch/scrubbed/samwhite/20180129_trimmed_again/NR019_S7_L002_R2_001_val_2_val_2.fastq i1 /gscratch/scrubbed/samwhite/20180129_trimmed_again/NR021_S8_L001_R1_001_val_1_val_1.fastq i2 /gscratch/scrubbed/samwhite/20180129_trimmed_again/NR021_S8_L001_R2_001_val_2_val_2.fastq i1 /gscratch/scrubbed/samwhite/20180129_trimmed_again/NR021_S8_L002_R1_001_val_1_val_1.fastq i2 /gscratch/scrubbed/samwhite/20180129_trimmed_again/NR021_S8_L002_R2_001_val_2_val_2.fastq 
Results

Output folder: 20180322_SparseAssembler_novaseq_geoduck/

This completed much more quickly than the previous run (kmer = 61). The previous assembly took ~10 days, while this assembly completed in ~4 days!

The primary output file of interest is this FASTA file:

In order to get a rough idea of how this assembly looks, I ran it through Quast Version: 4.5, 15ca3b9:

python software/quast-4.5/quast.py \
-t 16
/mnt/owl/Athaliana/20180322_SparseAssembler_novaseq_geoduck/Contigs.txt

Quast output folder: results_2018_03_27_08_25_52/

Here’re the stats on the assembly:

Quast output (text): results_2018_03_27_08_25_52/report.txt

Quast output (HTML):[results_2018_03_27_08_25_52/report.html]https://ift.tt/2uof9nh)

//

This is definitely a better assembly than the kmer = 61 assembly.

N50 = 1149

Also, there’s a single, large contig of 56,361bp, and 54 contigs > 25,000bp. This is good.

Admittedly, I’m a little surprised (and, disappointed) the N50 is as small as it is. However, we have a pretty decent assembly on our hands!

Since SparseAssembler seems to actually run (and, relatively quickly), I’m very tempted to just throw ALL of our geoduck data at it and see how it turns out…

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