Sam’s Notebook: Samples Received – Crassostrea virginica (Eastern oyster) tissue from Lotterhos Lab (Northeastern University)

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20181017_samples_recd.jpg

Sample sheet (Google Sheet):

Samples were stored in -80oC:

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Sam’s Notebook: qPCR – C.gigas primer and gDNA tests with 18s and EF1 primers

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The [qPCR I ran earlier today to check for residual gDNA in Ronit’s DNased RNA] turned out terribly, due to a combination of bad primers and, possibly, bad gDNA.

I tracked down some different primers for testing:

  • Cg_18s_1644_F (SRID 1168)
  • Cg_18s_1750_R (SRID 1169)
  • EF1_qPCR_5′ (SRID 309)
  • EF1_qPCR_3′ (SRID 310)

In addition to BB15 from 20090519, I decided to test out BB16 from 20090519 as a positive control.

Samples were run on Roberts Lab CFX Connect (BioRad). All samples were run in duplicate. See qPCR Report (Results section) for plate layout, cycling params, etc.

qPCR master mix calcs (Google Sheet):

Sam’s Notebook: qPCR – Ronit’s DNAsed C.gigas Ploidy/Dessication RNA with 18s primers

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After DNasing Ronit’s RNA earlier today, I needed to check for any residual gDNA.

Identified some old, old C.gigas 18s primers that should amplify gDNA:

  • gigas18s_fw (SRID 157)
  • gigas18s_rv (SRID 156)

Used some old C.gigas gDNA (BB15 from 20090519) as a positive control.

Samples were run on Roberts Lab CFX Connect (BioRad). All samples were run in duplicate. See qPCR Report (Results section) for plate layout, cycling params, etc.

qPCR master mix calcs (Google Sheet):

Sam’s Notebook: VCF Splitting with bcftools

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Steven asked for some help trying to split a VCF in to individual VCF files.

VCF file (15GB): SNP.TRSdp5g95FnDNAmaf05.vcf.gz

Skip to the Results section if you don’t want to read through the tials and tribulations of getting this to work.

Here’s an overview of how I managed to get this to work and what didn’t work.

Figured out the VCF file needed to be sorted, bgzipped (part of htslib), and indexed with tabix, due to the following error when initially trying to process with VCF file using bcftools:

[W::vcf_parse] contig '' is not defined in the header. (Quick workaround: index the file with tabix.)
Undefined tags in the header, cannot proceed in the sample subset mode.

So, I did that:

  • Sort and bgzip:
 cat SNP.TRSdp5g95FnDNAmaf05.vcf | \ awk '$1 ~ /^#/ {print $0;next} {print $0 | "sort -k1,1 -k2,2n"}' | \ bgzip --threads 20 > SNP.TRSdp5g95FnDNAmaf05.sorted.vcf.gz 
  • Index with tabix:
 tabix --preset vcf SNP.TRSdp5g95FnDNAmaf05.sorted.vcf.gz 

This produced a separate file:

  • SNP.TRSdp5g95FnDNAmaf05.sorted.vcf.gz.tbi.

It seems as though this file must exist in the same directory as the source VCF for it to be utilized, although no commands work directly with this index file.

Then, tried biostars solution, but produces an error

  #!/bin/bash for file in *.vcf.gz; do for sample in `bcftools query -l $file`; do bcftools view -c1 -Oz -s $sample -o ${file/.vcf*/.$sample.vcf.gz} $file done done  

Resulting error:

 [E::bcf_calc_ac] Incorrect AN/AC counts at NC_035780.1:26174 

And empty split VCF files…

Tried tabix on unsorted bgzipped file yields this error:

 [E::hts_idx_push] chromosome blocks not continuous 

Tried modified sort:

  cat SNP.TRSdp5g95FnDNAmaf05.vcf | \ awk '$1 ~ /^#/ {print $0;next} {print $0 | "sort -k1,1V -k2,2n"}' | \ bgzip --threads 20 > SNP.TRSdp5g95FnDNAmaf05.sorted.vcf.gz  

Produces this error:

 [E::bcf_calc_ac] Incorrect AN/AC counts at NC_035780.1:26174 

And empty split VCF files…

Changed to new version of “view” – trying “call” instead (it seems that bcftools view is deprecated?):

  #!/bin/bash for file in *.vcf.gz; do for sample in `bcftools query -l $file`; do bcftools call \ --consensus-caller \ --output-type z \ --threads 18 \ --samples $sample --output-file ${file/.vcf.gz/.$sample.vcf.gz} \ $file done done  

Still results in empty output files.

Based off of the repeated error about AN/AC counts, tried to fill AN/AC values…

  bcftools plugin fill-AN-AC SNP.TRSdp5g95FnDNAmaf05.sorted.vcf.gz \ --output-type z \ --threads 18 \ --output SNP.TRSdp5g95FnDNAmaf05.sorted.ANACfill.vcf.gz  

And, ran this code:

  #!/bin/bash for file in SNP.TRSdp5g95FnDNAmaf05.sorted.ANACfill.vcf.gz; do for sample in `bcftools query -l $file`; do bcftools call \ --consensus-caller \ --output-type z \ --threads 18 \ --samples $sample --output-file ${file/.vcf.gz/.$sample.vcf.gz} \ $file done done  

Still results in empty files…

Try original code again (expanded shortened arguments to improve readability):

  #!/bin/bash for file in SNP.TRSdp5g95FnDNAmaf05.sorted.ANACfill.vcf.gz; do for sample in `bcftools query -l $file`; do bcftools view \ --min-ac 1 \ --output-type z \ --samples $sample \ --output-file ${file/.vcf*/.$sample.vcf.gz} \ --threads 18 \ $file done done  

P.S. I realize the outermost for loop is not necessary, but it was faster/easier to just quickly modify the code from that Biostars solution.

Sam’s Notebook: DNase Treatment – Ronit’s C.gigas Ploiyd/Dessication Ctenidia RNA

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After quantifying Ronit’s RNA earlier today, I DNased them using the Turbo DNA-free Kit (Ambion), according to the manufacturer’s standard protocol.

Used 1000ng of RNA in a 50uL reaction in a 0.5mL thin-walled snap cap tube. Samples were mixed by finger flicking and then incubated 30mins @ 37oC in a PTC-200 thermal cylcer (MJ Research), without a heated lid.

DNase inactivation was performed (0.1 volumes of inactivation reagent; 5uL), pelleted, and supe transferred to new 1.7mL snap cap tube.

Samples were stored on ice in preparation for qPCR to test for residual gDNA.

DNase calculations are here:

Samples will be permanently stored here (Google Sheet):

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Sam’s Notebook: RNA Quantification – Ronit’s C.gigas Ploidy/Dessication RNA

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Last Friday, Ronit quantified 1:10 dilutions of the RNA I isolated on 20181003 and the RNA he finished isolating on 20181011, but two of the samples (D11-C, T10-C) were still too concentrated.

I made 1:20 dilutions (1uL RNA in 19uL 0.1% DEPC-treated H2O) and quantified them using the Roberts Lab Qubit 3.0, with the RNA HS assay. Used 1uL of the diluted RNA.

New photo at 47.76152, -122.85183666666667

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October 15, 2018 at 11:00AM
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