Nanodrop e5 samples

Nanodropping e5 deep dive DNA samples and WGBS libraries

I have been trying to create WGBS libraries with the Zymo Pico Methyl Seq kit for 15 e5 deep dive samples. I successfully prepped 9 samples but 6 samples (3 POC and 3 POR) are still failing despite several iterations.

  • 20240613 prep - used 25 ng of input DNA, 9 library amp cycles, 9 index amp cycles. 9 samples successfully prepped; 6 samples did not (3 POR, 3 POC).
  • 20240807 prep - tried to redo the 6 samples that failed. Used 25 ng of input DNA, 9 library amp cycles, 9 index amp cycles. New polymerase was used. All samples failed
  • 20240828 prep - tried to redo the 6 samples that failed. Used 25 ng of input DNA for POC and 10 ng input DNA for POR. 12 library amp cycles for POC, 9 library amp cycles for POR, 9 index amp cycles. All samples failed

I nanodropped the genomic DNA and all libraries on 9/5/24. See here for an explanation regarding nanodrop output and expectations.

Here are the results broken down by species.

Acropora pulchra

All Acropora samples successfully prepped on the first try hooray!

Number Species Type DateMade Qubit/TS_concentration_ng_ul Nanodrop_concentration_ng_ul A260 A280 260/280 260/230 NotebookPost Notes
413 Acropora pulchra Genomic DNA 20210902 19.9 11.6 0.233 0.082 2.85 3.98 https://github.com/Kterpis/Putnam_Lab_Notebook/blob/master/_posts/2021-09-02-20210902-RNA-DNA-extractions-from-E5-project.md  
413 Acropora pulchra Library 20240613 9.64 109.8 2.196 1.738 1.26 0.76 https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-06-13-Zymo-Pico-Methyl-Seq-Library-Prep.md  
423 Acropora pulchra Genomic DNA 20210903 17.7 13.4 0.267 0.104 2.57 1.8 https://github.com/Kterpis/Putnam_Lab_Notebook/blob/master/_posts/2021-09-03-20210903-RNA-DNA-extractions-from-E5-project.md Labeled as POR-75 in KXT extraction post
423 Acropora pulchra Library 20240613 8.61 99.3 1.987 1.401 1.42 0.95 https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-06-13-Zymo-Pico-Methyl-Seq-Library-Prep.md Labeled as POR-75 in KXT extraction post
427 Acropora pulchra Genomic DNA 20211012 19.6 9 0.179 0.056 3.2 -12.75 https://kterpis.github.io/Putnam_Lab_Notebook/20211012-RNA-DNA-extractions-from-E5-project/ Took 3 nanodrop readings, reported last one
427 Acropora pulchra Library 20240613 8.82 49.5 0.99 0.855 1.16 0.64 https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-06-13-Zymo-Pico-Methyl-Seq-Library-Prep.md  
439 Acropora pulchra Genomic DNA 20211102 19.9 27.2 0.543 0.254 2.14 4.46 https://kterpis.github.io/Putnam_Lab_Notebook/20211102-RNA-DNA-extractions-from-E5-project/  
439 Acropora pulchra Library 20240613 11.9 69.4 1.387 1.043 1.33 0.82 https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-06-13-Zymo-Pico-Methyl-Seq-Library-Prep.md  
467 Acropora pulchra Genomic DNA 20210921 21.9 42.6 0.852 0.419 2.04 3.26 https://github.com/Kterpis/Putnam_Lab_Notebook/blob/master/_posts/2021-09-21-20210921-RNA-DNA-extractions-from-E5-project.md DNA 1 and 2 Qubit readings are 27.6 and 27.4 but average is reported as 4.79 on github and on master sample sheet; JA re-qubited on 20240229 and got 21.9 ul/ng
467 Acropora pulchra Library 20240613 16 130.6 2.613 1.687 1.55 1.15 https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-06-13-Zymo-Pico-Methyl-Seq-Library-Prep.md DNA 1 and 2 Qubit readings are 27.6 and 27.4 but average is reported as 4.79 on github and on master sample sheet; JA re-qubited on 20240229 and got 21.9 ul/ng

Porites evermanni

471 and 491 were successful on the first try. 421, 487 and 489 have not been successful. The Nanodrop quality of the genomic DNA for all samples is pretty bad but 2 sampeles did successfully prep. Library quality is also pretty low.

Number Species Type DateMade Qubit/TS_concentration_ng_ul Nanodrop_concentration_ng_ul A260 A280 260/280 260/230 NotebookPost Notes
421 Porites evermanni Genomic DNA 20211008 3.41 -3.2 -0.06 -0.67 0.91 0.48 https://kterpis.github.io/Putnam_Lab_Notebook/20211008-RNA-DNA-extractions-from-E5-project/ Took 4 nanodrop readings, reported last one
421 Porites evermanni Library 20240613 NA 21.8 0.436 0.643 0.68 0.28 https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-06-13-Zymo-Pico-Methyl-Seq-Library-Prep.md  
421 Porites evermanni Library 20240807 0.116 22.9 0.458 0.654 0.7 0.32 https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-08-07-Zymo-Pico-Methyl-Seq-Library-Prep.md  
421 Porites evermanni Library 20240828 NA 15.6 0.311 0.492 0.63 0.27 https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-08-28-Zymo-Pico-Methyl-Seq-Library-Prep.md Input is 10 ng
471 Porites evermanni Genomic DNA 20211015 2.7 -2.7 -0.055 -0.063 0.87 0.48 https://kterpis.github.io/Putnam_Lab_Notebook/20211015-RNA-DNA-extractions-from-E5-project/  
471 Porites evermanni Library 20240613 5.25 38.4 0.769 0.69 1.11 0.6 https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-06-13-Zymo-Pico-Methyl-Seq-Library-Prep.md  
487 Porites evermanni Genomic DNA 20211122 2.31 -1.7 -0.033 -0.049 0.67 0.38 https://github.com/Kterpis/Putnam_Lab_Notebook/blob/master/_posts/2021-11-22-20211122-RNA-DNA-extractions-from-E5-project.md Took 3 nanodrop readings, reported last one
487 Porites evermanni Library 20240613 NA 14.4 0.289 0.435 0.66 0.27 https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-06-13-Zymo-Pico-Methyl-Seq-Library-Prep.md  
487 Porites evermanni Library 20240807 0.139 26.1 0.523 0.745 0.7 0.31 https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-08-07-Zymo-Pico-Methyl-Seq-Library-Prep.md  
487 Porites evermanni Library 20240828 0.213 13.5 0.271 0.469 0.58 0.24 https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-08-28-Zymo-Pico-Methyl-Seq-Library-Prep.md Input is 10 ng
489 Porites evermanni Genomic DNA 20211129 3.72 -2.4 -0.048 -0.07 0.68 0.99 https://github.com/Kterpis/Putnam_Lab_Notebook/blob/master/_posts/2021-11-29-20211129-RNA-DNA-extractions-from-E5-project.md Took 3 nanodrop readings, reported last one
489 Porites evermanni Library 20240613 NA 15.8 0.315 0.534 0.59 0.23 https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-06-13-Zymo-Pico-Methyl-Seq-Library-Prep.md  
489 Porites evermanni Library 20240807 0.204 18.4 0.367 0.529 0.69 0.3 https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-08-07-Zymo-Pico-Methyl-Seq-Library-Prep.md  
489 Porites evermanni Library 20240828 0.144 28.5 0.571 0.705 0.81 0.37 https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-08-28-Zymo-Pico-Methyl-Seq-Library-Prep.md Input is 10 ng
491 Porites evermanni Genomic DNA 20211101 2.11 -3.1 -0.061 -0.07 0.88 -1.89 https://kterpis.github.io/Putnam_Lab_Notebook/20211101-RNA-DNA-extractions-from-E5-project/ Took 3 nanodrop readings, reported last one
491 Porites evermanni Library 20240613 4.65 15.7 0.313 0.398 0.79 0.33 https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-06-13-Zymo-Pico-Methyl-Seq-Library-Prep.md  

Pocillopora tuahiniensis

403 and 417 were successful on the first try. 385, 393 and 401 have not been successful. The Nanodrop quality of the genomic DNA for all samples is decent so I am surprised that three of the libraries are not amplifying. I may just need to increase the number of amp and index cycles, as I have gotten a little bit of amplification from the POC samples but not enough for sequencing.

Number Species Type DateMade Qubit/TS_concentration_ng_ul Nanodrop_concentration_ng_ul A260 A280 260/280 260/230 NotebookPost Notes
385 Pocillopora tuahiniensis Genomic DNA 20211012 25.5 29.1 0.581 0.284 2.05 3.34 https://kterpis.github.io/Putnam_Lab_Notebook/20211012-RNA-DNA-extractions-from-E5-project/ Took 3 nanodrop readings, reported last one
385 Pocillopora tuahiniensis Library 20240613 NA 9.7 0.195 0.324 0.6 0.23 https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-06-13-Zymo-Pico-Methyl-Seq-Library-Prep.md  
385 Pocillopora tuahiniensis Library 20240807 1.69 36.7 0.733 0.828 0.89 0.44 https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-08-07-Zymo-Pico-Methyl-Seq-Library-Prep.md  
385 Pocillopora tuahiniensis Library 20240828 0.236 15.5 0.309 0.499 0.62 0.26 https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-08-28-Zymo-Pico-Methyl-Seq-Library-Prep.md Increasing number of cycles in Section 4 from 9 to 12
393 Pocillopora tuahiniensis Genomic DNA 20210903 29.4 10.5 0.21 0.071 2.97 -7.88 https://github.com/Kterpis/Putnam_Lab_Notebook/blob/master/_posts/2021-09-03-20210903-RNA-DNA-extractions-from-E5-project.md Took 4 nanodrop readings, reported last one
393 Pocillopora tuahiniensis Library 20240613 NA 9.3 0.186 0.33 0.56 0.21 https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-06-13-Zymo-Pico-Methyl-Seq-Library-Prep.md Labeled as POC-43 in KXT extraction post
393 Pocillopora tuahiniensis Library 20240807 NA 17 0.339 0.524 0.65 0.27 https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-08-07-Zymo-Pico-Methyl-Seq-Library-Prep.md Labeled as POC-43 in KXT extraction post
393 Pocillopora tuahiniensis Library 20240828 0.151 16.3 0.326 0.512 0.64 0.26 https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-08-28-Zymo-Pico-Methyl-Seq-Library-Prep.md Labeled as POC-43 in KXT extraction post; Increasing number of cycles in Section 4 from 9 to 12
401 Pocillopora tuahiniensis Genomic DNA 20211118 30.8 26.3 0.527 0.245 2.15 3.48 https://kterpis.github.io/Putnam_Lab_Notebook/20211118-RNA-DNA-extractions-from-E5-project/  
401 Pocillopora tuahiniensis Library 20240613 0.05 10.9 0.218 0.354 0.62 0.25 https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-06-13-Zymo-Pico-Methyl-Seq-Library-Prep.md  
401 Pocillopora tuahiniensis Library 20240807 3.82 37.3 0.747 0.804 0.93 0.46 https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-08-07-Zymo-Pico-Methyl-Seq-Library-Prep.md  
401 Pocillopora tuahiniensis Library 20240828 0.207 15.5 0.31 0.512 0.61 0.26 https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-08-28-Zymo-Pico-Methyl-Seq-Library-Prep.md Increasing number of cycles in Section 4 from 9 to 12
403 Pocillopora tuahiniensis Genomic DNA 20211129 29.1 45.4 0.908 0.455 2 1.21 https://github.com/Kterpis/Putnam_Lab_Notebook/blob/master/_posts/2021-11-29-20211129-RNA-DNA-extractions-from-E5-project.md Labeled as POC-55 in KXT extraction post
403 Pocillopora tuahiniensis Library 20240613 8.67 86.5 1.731 1.194 1.45 0.97 https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-06-13-Zymo-Pico-Methyl-Seq-Library-Prep.md Labeled as POC-55 in KXT extraction post
417 Pocillopora tuahiniensis Genomic DNA 20211020 51.4 108.9 2.178 1.128 1.93 2.45 https://kterpis.github.io/Putnam_Lab_Notebook/20211020-RNA-DNA-extractions-from-E5-project/ Labeled as POC-238 in KXT extraction post
417 Pocillopora tuahiniensis Library 20240613 9.3 158.7 3.174 2.141 1.48 1.05 https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-06-13-Zymo-Pico-Methyl-Seq-Library-Prep.md Labeled as POC-238 in KXT extraction post

For all samples/species, quality of genomic DNA is higher than library quality, which makes sense to me since the library prep process probably degrades the nucleic acid in some ways.

Written on September 5, 2024