Tag Archives: library prep

Library Quality Assessment – C.gigas OA larvae Illumina libraries

Ran the 400ppm library and the 1000ppm library preps on a DNA1000 Assay Chip (Agilent) on the Agilent 2100 Bioanalyzer.

 

Results:

Data File (XAD): 2100_expert_DNA_1000_DE72902486_2015-03-02_09-18-02.xad

Electropherogram overlay of both samples:

Red = 400ppm

Blue = 1000ppm

 

 

 

Measurement data and parameters are here: 20150302_Bioanalyzer_Cgigas_400_1000ppm_BS-Seq

 

Both libraries look good; no adaptor contamination (peak would be present at ~125bp), good library sizes.

Pooled equal quantities of each library, based off the concentration values above, to prepare the sample for sequencing.

Component Volume (μL) Quantity (ng)
400ppm library 10 14.7
1000ppm library 1.09 14.7
Buffer EB 7.81 N/A
1% Tween20 2.1 N/A
Total 21 N/A

 

The pooled libraries will be submitted tomorrow to the Genomics Core Facility at the Univ. of Oregon for high-throughput sequencing (100bp, SE) on the HiSeq2500 (Illumina). Sample order #2212.

Share

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.

Share

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…

Share

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.

 

Share

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.

Share

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.

 

Share

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
Share

Library Cleanup – LSU C.virginica MBD BS Library

I was contacted by the sequencing facility at the University of Oregon regarding a sample quality issue with our library.  As evidenced by the electropherogram below, there is a great deal of adaptor primer dimer (the peak at 128bp):

 

This is a problem because such a high quantity of adaptor sequence will result in the majority of reads coming off the Illumina being just adaptor sequences.

With the remainder of the library sample prepared earlier, I performed the recommended clean up procedure for removing adaptor sequences in the EpiNext Post-Bisulfite DNA Library Preparation Kit – Illumina (Epigentek).    Briefly:

  • Brought sample volume up to 20uL with NanoPure H2O (added 9.99uL)

  • Added equal volume of MQ Beads

  • Washed beads 3x w/80% EtOH

  • Eluted DNA w/12uL Buffer EB (Qiagen)

After clean up, quantified the sample via fluorescence using the Quant-iT DNA BR Kit (Life Technologies/Invitrogen).  Used 1uL of the sample and the standards.  All standards were run in duplicate and read on a FLx800 plate reader (BioTek).

Results are here: 20150122 – LSU_virginicaMBDlibraryCleanup

Library concentration = 2.46ng/uL

Brought the entire sample up to 20uL with Buffer EB (Qiagen) and a final concentration of 0.1% Tween-20 (required by the sequencing facility).

Sent sample to the University of Oregon to replace our previous submission.

Share

Bisulfite NGS Library Prep – LSU C.virginica Oil Spill MBD Bisulfite DNA and Emma’s C.gigas Larvae OA Bisulfite DNA (continued from yesterday)

Continued library prep from yesterday. Set up Library Amplification according to the protocol. The samples received the following Barcode Indices:

  • HB2 – 1 (ATCACG)
  • HB5 – 2 (CGATGT)
  • HB16 – 3 (TTAGGC)
  • HB30 – 4 (TGACCA)
  • NB3 – 5 (ACAGTG)
  • NB6 – 6 (GCCAAT)
  • NB11 – 7 (CAGATC)
  • NB21 – 12 (CTTGTA)
  • 1A1 – 2 (CGATGT)
  • 1A2 – 1 (ATCACG)
  • 6A1 – 4 (TGACCA)
  • 6A2 – 5 (ACAGTG)
  • 103B1 – 6 (GCCAAT)
  • 103B2 – 7 (CAGATC)
  • 105A4 – 12 (CTTGTA)
  • 105A5 – 11 (GGCTAC)

Due to differences in input DNA quantities, samples were run with different numbers of thermal cycles.

13 thermal cycles were run for the following samples:

  • 1A1
  • 105A4
  • 105A5

22 thermal cycles were run for the following samples:

  • HB2
  • HB5
  • HB16
  • HB30
  • NB3
  • NB6
  • NB11
  • NB21
  • 1A2
  • 6A1
  • 6A2
  • 103B1
  • 103B2

Samples were quantified with 1uL of each sample using the Quant-iT dsDNA BR Kit (Invitrogen). Used 5uL of each standard and standards were run in duplicate.

Results:

Share

Bisulfite NGS Library Prep – LSU C.virginica Oil Spill Bisulfite DNA and Emma’s C.gigas Larvae OA Bisulfite DNA

Constructed next generation libraries (Illumina) using the bisulfite-treated DNA from yesterday using the EpiNext Post-Bisulfite DNA Library Preparation Kit – Illumina (Epigentek). Samples were processed according to the manufacturer’s protocol up to Section 8 (Library Amplification) with the following changes:

– Skipped Section 7.1 (recommended to do so in the protocol due to low quantity of input DNA)

Samples were stored O/N @ -20C.

dA Tailing Master Mix

10x Tailing Buffer 1.5uL x 17.6 = 26.4uL

Klenow 1uL x 17.6 = 17.6uL

H2O 0.5uL x 17.6 = 8.8uL

Add 3uL of master mix to each sample

Adaptor Ligation

2x Ligation Buffer 17uL x 17.6 – 299.2uL

T4 DNA Ligase 1uL x 17.6uL = 17.6uL

Adaptors 1uL x 17.6 = 17.6uL

Added 19uL of master mix to each sample

dsDNA Conversion Master Mix

5x Conversion Buffer 4uL x 17.6 = 70.4uL

C.P. 2uL x 17.6 = 35.2uL

H2O 3uL x 18.6 = 52.8uL

Add 9uL of master mix to each sample

End Repair

10x Buffer 2uL x 17.6 = 35.2uL

Enzyme 1uL x 17.6 = 17.6uL

H2O 5uL x 17.6 = 88uL

Added 8uL of master mix to each sample

Share