Category Archives: Crassostrea gigas larvae OA (2011) bisulfite sequencing

BS-seq Library Prep – C.gigas Larvae OA 1000ppm

Bisulfite Conversion

Pooled 200ng each of the sheared 1B1 (4μL) & 1B2 (used the entire sample, 20μL) 5.13.11 1000ppm C.gigas larvae DNA samples for a total of 400ng. Total volume = 24μL.

Quantified the pooled DNA using the NanoDrop1000 (ThermoFisher) prior to initiating bisulfite conversion.

Clearly, the NanoDrop measurements differ from the expected concentration. NanoDrop suggests the total amount of input DNA is ~1400ng (58ng/μL x 24μL = 1392ng). This is most likely due to RNA carryover, as DNA quantification using a fluorescence-based, double-stranded DNA assay performed previously shows a drastically lower concentration.

Proceeded with bisulfite conversion using the Methylamp DNA Modification Kit (Epigentek) in 1.5mL tube, according to the manufacturer’s protocol:

  • Added 1μL to sample, incubated 10mins @ 37C in water bath
  • Made fresh R1/R2/R3 solution (1.1mL R3 buffer added to vial of R2, vortexed 2mins, 40μL R1 added to mixture – Remainder stored @ -20C in “-20C Kit Components Box”)
  • Added 125μL of R1/R2/R3 solution to sample, incubated 90mins @ 65C in heating block with water
  • Addd 300μL R4 to sample, mixed, transferred to column, spun 12,000RPM 30s
  • Added 200μL R5 to column, spun 12,000RPM 30s
  • Added 50μL R1/ethanol solution to column, incubated 8mins @ RT, spun 12,000RPM 30s
  • Washed column with 200μL of 90% EtOH, spun 12,000RPM 30s; repeated one time.
  • Eluted DNA with 12μL R6, spun 12,000RPM 30s

Quantified post-bisulfite-treated sample on NanoDrop1000:

Definitely a low yield (~108ng) relative to the input (~400ng). Will proceed with Illumina library prep.

 

Library Prep

Illumina library prep was performed with EpiNext Post-Bisulfite DNA Library Preparation Kit (Illumina) (Epigentek).  Changes to the manufacturer’s protocol:

  • Samples were transferred to 1.5mL snap cap tubes for all magnetic bead steps in order to fit in our tube magnets.
  • PCR cycles: 15

No other changes were made to the manufacturer’s protocol.

Epigentek Barcode Indices assigned, per their recommendations for using two libraries for multiplexing (this will be combined with the 400ppm library):

Barcode #12 – CTTGTA

The library was stored @ -20C and will be checked via Bioanalyzer on Monday.

DNA Quantification – C.gigas Larvae 1000ppm

After the discovery that there wasn’t any DNA in the BS-seq Illumina library prep and no DNA in the bisulfite-treated DNA pool, I decided to try to recover any residual DNA left in the 1B2 sample. Sample 1B2 (sheared on 20150109) was dry, so I added 20μL of Buffer EB (Qiagen) to the tube. I vortexed both the 1B1 and 1B2 samples and quantified on the NanoDrop1000 (ThermoFisher). I also re-quantified the pooled BS-treated sample that had been used as input DNA for the libraries.

Results:

Spreadsheet: 20150226_Claire_sheared_Emma_1000ppm_OD260s

Sample 1B1 has ample DNA in it. Since these samples are pools of larvae, we may be able to just proceed with this sample and not worry about pooling with the biological replicate 1B2.

Sample 1B2 has a low amount of DNA, but it’s a usable quantity (total 400ng).

Pooled samples has nothing.

Will make a new pool of DNA from both 1B1 and 1B2 and attempt to make a new bisulfite-treated library.

Library Prep – Quantification of C.gigas larvae OA 1000ppm library

The completed BS Illumina library made on Friday (1000ppm) was quantified via fluorescence using the Quant-iT DNA BR Kit (Life Technologies/Invitrogen).  Also quantified Jake’s libraries. Used 1μL of  each sample and the standards.  All standards were run in duplicate.  Due to limited sample, the libraries were only processed singularly, without replication.  Fluorescence was measured on a FLx800 plate reader (BioTek), using the Gen5 (BioTek) software for all calculations.

Results:

20150209_CgigasOA_BSlibrraryQuants_OluridaLibraryQuants

The good news is that the standard curve looked fine, with an R²=0.998.

The bad news is that there’s no detectable DNA in the sample, just like last time.

Possibly something is totally shot with this sample?  Will quantify the sheared DNA and decide what to do.

I quantified the sheared DNA and there’s nothing there! Where did it go? I just don’t get it. It was sheared, speed-vac’d and resuspended.  All the DNA should still be in the tubes…

Bisulfite NGS Library Prep – Bisulfite Conversion & Illumina Library Construction of C.gigas larvae DNA

Bisulfite Conversion

The previous attempt at constructing a library for the 1000ppm larvae samples failed. I had previously sheared, quantified, and concentrated the DNA from this sample. As I had done previously, I combined 50ng from each of the two 1000ppm samples for a total of 100ng, and brought the sample volume up to 24μL with NanoPure H2O.

Bisulfite conversion was performed with the Methylamp DNA Modification Kit (Epigentek) according to the manufacturer’s protocol.

Sample was eluted with 10μL of Buffer R6 for immediate use.

 

Library Prep

Bisulfite Illumina library was made with EpiNext Post-Bisulfite DNA Library Preparation Kit (Illumina) (Epigentek).  Changes to the manufacturer’s protocol:

  • Samples were transferred to 1.5mL snap cap tubes for all magnetic bead steps in order to fit in our tube magnets.
  • Skipped Step 7.1 (per manufacturer’s recommendation for samples starting with <200ng)
  • Ran 13 cycles during the library amplification step (per manufacturer’s recommendation for samples starting with 100ng)

Sample was transferred to 1.5mL snap cap tube and stored @ -20C.  Will quantify library on Monday when Jake is also finished with his 12 libraries.

 

Bisuflite NGS Library Prep – C.gigas larvae OA bisulfite library quantification

The two completed BS Illumina libraries (400ppm and 1000ppm) were quantified via fluorescence using the Quant-iT DNA BR Kit (Life Technologies/Invitrogen).  Used 1uL of  each sample and the standards.  All standards were run in triplicate.  Due to limited sample, the two libraries were only processed singularly, without replication.  Fluorescence was measured on a FLx800 plate reader (BioTek).

 

Results:

The standard curve, raw fluorescence, and calculated concentrations (as determined by the Gen5 (BioTek) software) can be seen here: 20150128_CgigasOA_BSlibrraryQuants_OluridaLibraryQuants

The standard curve was excellent, exhibiting a R² value = 0.999

 

Sample Concentration (ng/uL)
400ppm 10.592
1000ppm 0.0

 

The 400ppm library looks great, with a good yield.

The 1000ppm library appears to have no measurable quantity of DNA in it.  This is surprising, and disconcerting, as both samples were processed in parallel.  As such, there should be virtually no difference between them, in regards to the library construction process and subsequent yields.

To verify that this wasn’t a pipetting error on my part, I re-quantified the 1000ppm library (in duplicate) and still no detectable DNA.

Will repeat the bisulfite conversion and library construction process on the 1000ppm sample in order to generate a usable library for sequencing.

Bisuflite NGS Library Prep – C.gigas larvae OA bisulfite DNA (continued from yesterday)

Continued Illumina library prep of bisulfite-treated DNA samples (400ppm and 1000ppm; from 20150114)  with Methylamp DNA Modification Kit (Epigentek). Performed bead clean up immediately after End Repair.

PCR cycles: 14

No other changes were made to the manufacturer’s protocol.

Epigentek Barcode Indices assigned, per their recommendations for using two libraries for multiplexing:

400ppm – barcode #6 – GCCAAT

1000ppm – barcode #12 – CTTGTA

The two libraries were stored @ -20C and will be quantified tomorrow.

 

Bisuflite NGS Library Prep – C.gigas larvae OA bisulfite DNA

The two pooled bisulfite-treated DNA samples (400ppm and 1000ppm) from 20150114 were used to prepare bisulfite Illumina libraries with EpiNext Post-Bisulfite DNA Library Preparation Kit (Illumina) (Epigentek).  Changes to the manufacturer’s protocol:

  • Samples were transferred to 1.5mL snap cap tubes for all magnetic bead steps in order to fit in our tube magnets.
  • Stopped after End Repair step (prior to magnetic bead clean up).  Samples stored @ -20C

DNA Bisulfite Conversion – C.gigas larvae OA Sheared DNA

After discussing with Steven, decided to use only samples from 20110513, due to high algae amounts present in the 20110519 samples.

Pooled the DNA of the 400ppm samples (6B2 & 6B5) and pooled the DNA of the 1000ppm samples(1B1 & 1B2) , for a total of two samples. 50ng of each sample was used to make the pools, for a toal of 100ng of DNA in each pool. Calculations are below:
https://docs.google.com/spreadsheets/d/1e7EF05akWeBtO7Xz0UWXhIzRlkVU5HA_rjCE3c4SPEw/edit?usp=sharing

Each pooled sample was brought up to a final volume of 24μL for use in bisulfite conversion kit.

Performed bisulfite conversion of sheared DNA from 20150113 using the Methylamp DNA Modification Kit (Epigentek) according the manufacturer’s protocol.

Samples were eluted with 10μL of Buffer R6 and stored @ -20C.

 

SpeedVac – C.gigas larvae OA DNA

The DNA extracted and sheared on 20150109 was in a volume too great to proceed with bisulfite conversion.  Dried the samples to complete dryness in a SpeedVac (~4hrs).  Reconsitituted DNA in 24μL of NanoPure water.  Will bisulfite convert tomorrow.

Sample list:

SAMPLE ID DATE TREATMENT (ppm) # LARVAE TOTAL DNA (ng)
6B5 20110513 400 5,000 105
1B2 20110513 1000 5,000 109
6B2 20110513 400 10,000 117
1B1 20110513 1000 10,000 593
1B1 20110519 1000 NA 500
1B2 20110519 1000 NA 500
6B2 20110519 400 NA 269
6B1 20110519 400 NA 345

 

Updated the quantification spreadsheet with a column labeled “[New](ug/uL)” that calculates the new concentrations of the above samples in the 24μL volume.

https://docs.google.com/spreadsheets/d/1e7EF05akWeBtO7Xz0UWXhIzRlkVU5HA_rjCE3c4SPEw/edit?usp=sharing

 

DNA Isolation – C.gigas larvae from 2011 NOAA OA Experiment

DNA was isolated from the following samples:

SAMPLE ID DATE TREATMENT (ppm) # LARVAE
6B5 20110513 400 5,000
1B2 20110513 1000 5,000
6B2 20110513 400 10,000
1B1 20110513 1000 10,000
1B1 20110519 1000 NA
1B2 20110519 1000 NA
6B2 20110519 400 NA
6B1 20110519 400 NA

 

Some tubes contained a high quantity of algae, based on quantity of material in tube and overall green color.

Samples 1B1 & 1B2 from 20110519 have excessive quantities of algae.

Samples 6B1 & 6B1 from 20110519 have a fair amount of algae.

See pic:

 

Sample tubes after brief spin, prior to DNA isolation.

Prior to isolation, samples were briefly spun (12,000g, 15s @ RT). Supernatants were discarded.

 

 

DNA Isolation

DNA was isolated using the DNeasy Blood & Tissue Kit (Qiagen).

Samples were resuspended in 180uL of Buffer AL and 20uL of Proteinase K. Samples were mixed by vortexing and incubated @ 56C O/N.

The manufacturer’s protocol (Purification of Total DNA from Animal Tissues (Spin-Column Protocol)) was followed.

Due to low quantities of starting tissue, samples were eluted with 200μL of Buffer EB to maximize DNA recovery.

 

DNA Quantification

Samples were prepared for quantification via fluorescence using the Quant-iT DNA BR Kit (Life Technologies/Invitrogen). The manufacturer’s protocol was altered to use 5μL of sample and 5μL of standards (instead of 10μL) in each well. All samples/standards were run in duplicate and read on a FLx800 plate reader (BioTek).

Mean fluorescence of the standards were plotted with a best-fit line. The resulting equation from the best-fit line was used to determine sample concentrations from their mean fluorescence.

 

Results:

Calcs and resulting quantities are here:

https://docs.google.com/spreadsheets/d/1e7EF05akWeBtO7Xz0UWXhIzRlkVU5HA_rjCE3c4SPEw/edit?usp=sharing

 

All samples have yields great enough to proceed with shearing and bisulfite conversion.

Samples 1B1 and 1B2 from 20110519 have extremely large yields.  This is not surprising, considering the amount of algae present in the source tubes.  Will process only 500ng from each sample.

 

 

DNA Shearing

Adjusted volume of all samples to 190μL using Buffer EB (Qiagen) in 1.5mL snap-cap tubes.

Samples were sonicated/sheared in the Bioruptor (Diagenode) with the following cycling protocol:

25 cycles of:

30s on

30s off

Cycling params were adjusted from the last time I performed this, since I felt the final sheared size was a bit on the small size.

After shearing, samples were stored @ 4C until I could SpeedVac them to reduce their volumes, as the bisulfite treatment step requires volumes < 24uL.