Tag Archives: coral

Data Received – Jay’s Coral epiRADseq – Not Demultiplexed

Previously downloaded Jay’s epiRADseq data that was provided by the Genomic Sequencing Laboratory at UC-Berkeley. It was provided already demultiplexed (which is very nice of them!). To be completionists on our end, we requested the non-demultiplexed data set.

Downloaded the FASTQ files from the project directory to Owl/nightingales/Porites_spp:

time wget -r -np -nc --ask-password ftp://gslftp@gslserver.qb3.berkeley.edu/160830_100PE_HS4KB_L4

 

It took awhile:

FINISHED --2016-09-19 11:39:21--
Total wall clock time: 4h 26m 21s
Downloaded: 11 files, 36G in 4h 17m 18s (2.39 MB/s)

Here are the files:

  • JD001_A_S1_L004_R2_001.fastq.gz
  • JD001_A_S1_L004_R1_001.fastq.gz
  • JD001_A_S1_L004_I1_001.fastq.gz
  • 160830_100PE_HS4KB_L4_Stats/
    • AdapterTrimming.txt
    • ConversionStats.xml
    • DemultiplexingStats.xml
    • DemuxSummaryF1L4.txt
    • FastqSummaryF1L4.txt

 

Generated MD5 checksums for each file:

for i in *.gz; do md5 $i >> checksums.md5; done

 

 

Calculate total number of reads for this sequencing run:

totalreads=0; for i in *S1*R*.gz; do linecount=`gunzip -c "$i" | wc -l`; readcount=$((linecount/4)); totalreads=$((readcount+totalreads)); done; echo $totalreads

Total reads: 662,868,166 (this isn’t entirely accurate, as it is counting all three files; probably should’ve just counted the R1 and R2 files…)

 

 

Calculate read counts for each file and write the data to the readme.md file in the Owl/web/nightingales/Porites_spp directory:

for i in *S1*R*.gz; do linecount=`gunzip -c "$i" | wc -l`; readcount=$(($linecount/4)); printf "%st%sn" "$i" "$readcount" >> readme.md; done

 

See this Jupyter notebook for code explanations.

 

Added sequencing info to Next_Gen_Seq_Library_Database (Google Sheet) and the Nightingales Spreadsheet (Google Sheet) and Nightingales Fusion Table (Google Fusion Table).

Share

Data Received – Jay’s Coral epiRADseq

We received notice that Jay’s coral (Porites spp) epiRADseq data was available from the Genomic Sequencing Laboratory at UC-Berkeley.

Downloaded the FASTQ files from the project directory to Owl/nightingales/Porites_spp:

time wget -r -np -nc -A "*.gz" --ask-password ftp://gslftp@gslserver.qb3.berkeley.edu/160830_100PE_HS4KB/Roberts

 

Generated MD5 checksums for each file:

for i in *.gz; do md5 $i >> checksums.md5; done

 

 

Calculate total number of reads for this sequencing run:

totalreads=0; for i in *.gz; do linecount=`gunzip -c "$i" | wc -l`; readcount=$((linecount/4)); totalreads=$((readcount+totalreads)); done; echo $totalreads

Total reads: 573,378,864

 

 

Calculate read counts for each file and write the data to the readme.md file in the Owl/web/nightingales/Porites_spp directory:

for i in *.gz; do linecount=`gunzip -c "$i" | wc -l`; readcount=$(($linecount/4)); printf "%st%sn" "$i" "$readcount" >> readme.md; done

 

See this Jupyter notebook for code explanations.

 

Added sequencing info to Next_Gen_Seq_Library_Database (Google Sheet) and the Nightingales Spreadsheet (Google Sheet) and Nightingales Fusion Table (Google Fusion Table).

Share

DNA Methylation Quantification – Coral DNA from Jose M. Eirin-Lopez (Florida International University)

Ran the coral DNA I quantified on 20160630 through the MethylFlash Methylated DNA Quantification Kit [Colorimetric] (Epigentek) kit to quantify global methylation.

Used 100ng of DNA per 8uL per replicate (x2 replicates = total 200ng in 16uL). Calcs are here (Google Sheet): 20160705_coral_DNA_methylation_calcs

Manufacturer’s protocol was followed.

Dilutions of kit reagents:

ME5 (1:1000) 2.6uL ME5 + 2597.4uL diluted ME1

ME6 (1:2000) 1.3uL ME6 + 2598.7uL diluted ME1

ME7 (1:5000) 0.52uL ME7 + 2599.48uL diluted ME1

Samples were quantified on the Seeb’s plate reader @ 450nm  (Wallac 1420 Victor 2  [Perkin Elmer])

Results:

Google Sheet: 20160707_coral_DNA_methylflash

sample treatment 5-mC(ng)
H1_1 nitrogen 0.8712248853
H1_10 nitrogen 0.6917168368
H1_12 control 0.2738478893
H1_5 nitrogen & phosphorous 0.9663585942
H1_6 control 0.6494783825
H1_8 nitrogen & phosphorous 0.4244913398
H24_1 nitrogen 0.372603297
H24_10 nitrogen 0.4237237786
H24_12 control 0.5350511937
H24_5 nitrogen & phosphorous 0.1495527697
H24_6 control 0.2291900804
H24_8 nitrogen & phosphorous 0.2213437801
H5_1 nitrogen -0.1233169902
H5_10 nitrogen 0.6997668774
H5_12 control 0.2307000493
H5_5 nitrogen & phosphorous -0.07790933048
H5_6 control 0.4562401662
H5_8 nitrogen & phosphorous 0.5949647121

 

Overall, it’s difficult to really interpret these results. I believe the data is a time course (e.g. H5 = hour 5, H24 = hour 24). Additionally, looking at treatments, there appear to be replicates, but it’s not clear what type of replicates they are (i.e. technical or biological). Generally, it seems like the control samples have lower quantities of methylated DNA than the treated samples. However, this doesn’t hold true for all three of the groups.

And, not that it really matters, but I don’t even know what species this is…

In any case, this was an attempt to gather some preliminary data for a grant that Steven is attempting to put together, so the original experiment and the subsequent data aren’t as robust as one would expect for a full-blown research project.

Share

DNA Quantification – Coral DNA from Jose M. Eirin-Lopez (Florida International University)

Quantified the DNA we received from Jose on 20160615 using the Qubit 3.0 Flouorometer (ThermoFisher) with the dsDNA Broad Range (BR) Kit according to the manufacturer’s protocol. Used 1μL of each sample.

Results are here (Google Sheet): Coral_DNA_QubitData_2016-06-30_08-45-56.xls

Here is a table of sample concentrations:

Sample Concentration(ng/μL)
H1 1 52.4
H1 5 34
H1 6 13
H1 8 22
H1 10 39
H1 12 52.4
H5 1 14.7
H5 5 20.8
H5 6 54
H5 8 18.4
H5 10 46.6
H5 12 29.8
H24 1 16.2
H24 5 25
H24 6 20.2
H24 8 22
H24 10 22
H24 12 30.6

 

Will proceed with DNA methylation assessment.

Share

Samples Received – Coral DNA from Jose M. Eirin-Lopez (Florida International University)

Steven received these coral DNA samples today. Here’s his post on Google Plus (stored @ 4C in FTR 213):

 

 

Here’s the email from Jose describing the samples:

“Dear Steven, the coral DNA samples were sent today by my student Javier (cc’ed here) to your lab. Here’s an excel attached with info for the samples including concentration and treatment of the coral from which they were extracted (N, nitrogen; NP, nitrogen+phosphorous; C, control).

Please let us know when you get these in the lab so we know all is fine!

thanks!

Chema”

Here’s the spreadsheet he sent (renamed for easier identification later on – original file sent was title DNA Qbit readings), uploaded to Google Drive:

20160615_coral_DNA_Qbit_readings.xls

Share