Tag Archives: Katie Lotterhos

Data Received – Bisulfite-treated Illumina Sequencing from Genewiz

Received notice the sequencing data was ready from Genewiz for the samples submitted 20151222.

Download the FASTQ files from Genewiz project directory:

wget -r -np -nc -A "*.gz" ftp://username:password@ftp2.genewiz.com/Project_BS1512183

Since two species were sequenced (C.gigas & O.lurida), the corresponding files are in the following locations:

http://owl.fish.washington.edu/nightingales/O_lurida/

http://owl.fish.washington.edu/nightingales/C_gigas/

 

In order to process the files, I needed to identify just the FASTQ files from this project and save the list of files to a bash variable called ‘bsseq':

bsseq=$(ls | grep '^[0-9]{1}_*' | grep -v "2bRAD")

Explanation:

bsseq=
  • This initializes a variable called “bsseq” to the values contained in the command following the equals sign.
$(ls | grep '^[0-9]{1}_*' | grep -v "2bRAD")
  • This lists (ls) all files, pipes them to the grep command (|), grep finds those files that begin with (^) one or two digits followed by an underscore ([0-9{1}_*), pipes those results (|) to another grep command which excludes (-v) any results containing the text “2bRAD”.

 

FILENAME SAMPLE NAME SPECIES
1_ATCACG_L001_R1_001.fastq.gz 1NF11 O.lurida
2_CGATGT_L001_R1_001.fastq.gz 1NF15 O.lurida
3_TTAGGC_L001_R1_001.fastq.gz 1NF16 O.lurida
4_TGACCA_L001_R1_001.fastq.gz 1NF17 O.lurida
5_ACAGTG_L001_R1_001.fastq.gz 2NF5 O.lurida
6_GCCAAT_L001_R1_001.fastq.gz 2NF6 O.lurida
7_CAGATC_L001_R1_001.fastq.gz 2NF7 O.lurida
8_ACTTGA_L001_R1_001.fastq.gz 2NF8 O.lurida
9_GATCAG_L001_R1_001.fastq.gz M2 C.gigas
10_TAGCTT_L001_R1_001.fastq.gz M3 C.gigas
11_GGCTAC_L001_R1_001.fastq.gz NF2_6 O.lurida
12_CTTGTA_L001_R1_001.fastq.gz NF_18 O.lurida

 

I wanted to add some information about the project to the readme file, like total number of sequencing reads generated and the number of reads in each FASTQ file.

Here’s how to count the total of all reads generated in this project

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

Total reads = 138,530,448

C.gigas reads: 22,249,631

O.lurida reads: 116,280,817

Code explanation:

totalreads=0;
  • Creates variable called “totalreads” and initializes value to 0.
for i in $bsseq;
  • Initiates a for loop to process the list of files stored in $bsseq variable. The FASTQ files have been compressed with gzip and end with the .gz extension.
do linecount=
  • Creates variable called “linecount” that stores the results of the following command:
`gunzip -c "$i" | wc -l`;
  • Unzips the files ($i) to stdout (-c) instead of actually uncompressing them. This is piped to the word count command, with the line flag (wc -l) to count the number of lines in the files.
readcount=$((linecount/4));
  • Divides the value stored in linecount by 4. This is because an entry for a single Illumina read comprises four lines. This value is stored in the “readcount” variable.
totalreads=$((readcount+totalreads));
  • Adds the readcount for the current file and adds the value to totalreads.
done;
  • End the for loop.
echo $totalreads
  • Prints the value of totalreads to the screen.

Next, I wanted to generate list of the FASTQ files and corresponding read counts, and append this information to the readme file.

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

Code explanation:

for i in $bsseq; do linecount=`gunzip -c "$i" | wc -l`; readcount=$(($linecount/4));
  • Same for loop as above that calculates the number of reads in each FASTQ file.
printf "%st%snn" "$i" "$readcount" >> readme.md;
  • This formats the the printed output. The “%st%snn” portion prints the value in $i as a string (%s), followed by a tab (t), followed by the value in $readcount as a string (%s), followed by two consecutive newlines (nn) to provide an empty line between the entries. See the readme file linked above to see how the output looks.
>> readme.md; done
  • This appends the result from each loop to the readme.md file and ends the for loop (done).

 

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Illumina Methylation Library Quantification – BS-seq Oly/C.gigas Libraries

Re-quantified the libraries that were completed yesterday using the Qubit3.0 dsDNA HS (high sensitivity) assay because the library concentrations were too low for the normal broad range kit.

Results:

Qubit Quants and Library Normalization Calcs: 20151222_qubit_illumina_methylation_libraries

SAMPLE CONCENTRATION (ng/μL)
1NF11 2.42
1NF15 1.88
1NF16 2.74
1NF17 2.54
2NF5 2.72
2NF6 2.44
2NF7 2.38
2NF8 1.88
M2 2.18
M3 2.56
NF2_6 2.5
NF_18 2.66

 

Things look pretty good. The TruSeq DNA Methylation Library Kit (Illumina) suggests that the libraries produced should end up with concentrations >3ng/μL, but we have plenty of DNA here to make a pool for running on the HiSeq2500.

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Bioanalyzer – Bisulfite-treated Oly/C.gigas DNA

Following the guidelines of the TruSeq DNA Methylation Library Prep Guide (Illumina), I ran 1μL of each sample on an RNA Pico 6000 chip on the Seeb Lab’s Bioanalyzer 2100 (Agilent) to confirm that bisulfite conversion from earlier today worked.

Results:

Data File 1(Bioanlyzer 2100): 2100 expert_Eukaryote Total RNA Pico_DE72902486_2015-12-18_21-05-04.xad

Data File 1(Bioanlyzer 2100): 2100 expert_Eukaryote Total RNA Pico_DE72902486_2015-12-18_21-42-55.xad

 

 

Firstly, the ladder failed to produce any peaks. Not sure why this happened. Possibly not denatured? Seems unlikely, but next time I run the Pico assay, I’ll denature the ladder aliquot I use prior to running.

Overall, the samples look as they should (see image from TruSeq DNA Methylation Kit manual below), albeit some are a bit lumpy.

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Bisulfite Treatment – Oly Reciprocal Transplant DNA & C.gigas Lotterhos DNA for BS-seq

After confirming that the DNA available for this project looked good, I performed bisulfite treatment on the following gDNA samples:

  • 1NF11
  • 1NF15
  • 1NF16
  • 1NF17
  • 2NF5
  • 2NF6
  • 2NF7
  • 2NF8
  • NF2_6
  • NF2_18
  • M2
  • M3

Sample names breakdown like this:

1NF#

1 = Fidalgo Bay outplants

NF = Fidalgo Bay broodstock origination

= Sample number

2NF#

Same as above, but:

2 = Oyster Bay outplants

NF2_# (Oysters grown in Oyster Bay; DNA provided by Katherine Silliman)

NF2 = Fidalgo Bay broodstock origination, family #2

= Sample number

M2/M3 = C.gigas from Katie Lotterhos

 

Followed the guidelines of the TruSeq DNA Methylation Library Prep Guide (Illumina).

Used the EZ DNA Methylation-Gold Kit (ZymoResearch) according to the manufacturer’s protocol with the following changes/notes:

  • Used 100ng DNA (per Illumina recs; Zymo recommends at least 200ng for “optimal results”).
  • Thermal cycling was performed in 0.5mL thin-wall tubes in a PTC-200 (MJ Research) using a heated lid
  • Centrifugations were performed at 13,000g
  • Desulphonation incubation for 20mins.

DNA quantity calculations are here (Google Sheet): 20151218_oly_bisulfite_calcs

Samples were stored @ -20C. Will check samples via Bioanalyzer before proceeding to library construction.

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