Do you have a Methods section for yeast whole genome sequencing?

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If you use our yeast Whole Genome Sequencing services, the following text can be used for the Methods section in scientific publications.  This method is applicable for Illumina short-read sequencing.

We follow this protocol very closely, although we may have to modify it slightly for specific issues that might arise in any particular sequencing project.  If you intend to use the verbiage below in a publication, we recommend that you contact us to discuss and review your specific project.  We can help you determine if the protocol is accurate for your project.

 

Methods

Illumina MiniSeq Short-Read Sequencing

Yeast Whole Genome Sequencing

  

DNA Extraction

The Zymo Research Quick-DNA Fungal/Bacterial Microprep Kit (Catalog No. D6007) was used to extract DNA from yeast cells. Yeast cells (10-20 mg wet weight) were resuspended in 200 μl PBS (phosphate buffered saline) and added to a ZR BashingBead™ Lysis Tube (0.1 & 0.5 mm) with 750 μL Lysis Solution. The tube was processed in a Qiagen (qiagen.com) TissueLyser LT bead beater at 50 Hz for 5 min. and centrifuged at 10,000 x g for 1 min. 400 μl supernatant was transferred to a Zymo-Spin™ IV Spin Filter in a Collection Tube and centrifuged at 7,000 x g for 1 min. To optimize performance, beta-mercaptoethanol was added to Genomic Lysis Buffer to a final dilution of 0.5% (v/v) or 500 μL / 100 mL. 1,200 uL Genomic Lysis Buffer was added to the filtrate, 800 μL of the mixture was transferred to a Zymo-Spin™ IC Column in a Collection Tube and centrifuged at 10,000 x g for 1 min. The flow-through was discarded and the previous step was repeated once. 200 uL DNA Pre-Wash Buffer was added to the Zymo-Spin™ IC Column and centrifuged at 10,000 x g for 1 min. 500 uL g-DNA Wash Buffer was added to the Zymo-Spin™ IC Column and centrifuged at 10,000 x g for 1 min. The Zymo-Spin™ IC Column was transferred to a 2 mL microcentrifuge tube and 20 μl of DNA Elution Buffer was added directly to the column matrix. After 3 min. incubation, the column was centrifuged at 10,000 x g for 30 seconds to elute ultra-pure DNA.

 

Library Prep

The Illumina (illumina.com) Nextera XT DNA Library Prep Kit was used to prepare extracted yeast DNA for sequencing.

 

Tagmentation

10 μl of TD and 5 μL of extracted gDNA were added to each plate well in a PCR plate and mixed by pipetting. 5 μl of ATM was added to each plate well, mixed by pipetting and the plate was centrifuged at 280 × g for 1 min. A predefined tagmentation program was run on an ABI GeneAmp PCR System 9700 thermal cycler. Immediately following its completion, 5 μl of NT was added to each plate well and mixed by pipetting. The plate was centrifuged at 280 × g for 1 min. followed by incubation at room temperature for 5 min.

 

Amplification and Indexing

Illumina Index 1 (i7) adapters and Index 2 (i5) adapters were uniquely assigned to each sample on an index adapter plate. 15 μl of NPM was added to each plate well and mixed by pipetting. The Index/NPM mixture was transferred to the PCR plate, mixed by pipetting, centrifuged at 280 × g for 1 min. and placed on the thermal cycler to run a predefined PCR program.

 

Library Cleanup

Library cleanup was performed using AMPure XP beads (beckman.com). The PCR product was centrifuged at 280 × g for 1 min. and 50 μL of product was transferred to a new PCR plate. 30 μL of AMPure XP beads was added to each plate well and the plate was shaken at 1800 rpm for 2 min. The plate was incubated at room temperature for 5 min. and placed on a magnetic stand until the liquid was clear. All supernatant was removed and discarded and the beads were washed twice with 200 μL 80% EtOH.  A 10 μL pipette was used to aspirate any residual alcohol. The plate was air-dried on a magnetic stand for 15 min. 52.5 μl of RSB was added to the beads and the plate was shaken at 1800 rpm for 2 min. The plate was incubated at room temperature for 2 min. and placed on a magnetic stand until the liquid was clear. 50 μL of supernatant was transferred to a new PCR plate.

 

Check Libraries

To check library size distribution, undiluted library was analyzed on an Agilent Technologies 2200 TapeStation using Agilent Technologies High Sensitivity D1000 ScreenTape.

 

Normalize Libraries

44 μL of LNA1 per sample and 8 uL of LNB1 per sample were added to a 15 mL conical tube. 45 μL of the LNA1/LNB1 mixture was added to each sample PCR plate well. The plate was shaken at 1800 rpm for 30 min. and placed on a magnetic stand until the liquid was clear. The supernatant was removed and discarded. The beads were washed twice with 45 μL of LNW1 and 30 μL of 0.1 N NaOH was added to each plate well. The plate was shaken at 1800 rpm for 5 min. After a 5-min. incubation, the samples were resuspended and shaken at 1800 rpm for 5 min. The plate was placed on a magnetic stand until the liquid was clear. The supernatant was transferred to a new PCR plate and the plate was centrifuged at 1000 × g for 1 min.

 

Pool, Denature, Dilute Libraries and Add PhiX Control

5 μL of each library was transferred from the PCR plate to a 2 mL microcentrifuge tube. A fresh dilution of 0.1 N NaOH was prepared by combining 900 μL of laboratory-grade water with 100 μL of stock 1.0 N NaOH. The denaturation process involved combining 5 μl of 1 nM library with 5 μL of 0.1 N NaOH, vortexing and incubating at room temperature for 5 min. 5 μL of 200 mM Tris-HCl, pH 7.0, was added to the denatured library, vortexed and centrifuged at 280 × g for 1 min. The denatured library was diluted with 985 μL Hybridization Buffer, vortexed and centrifuged at 280 × g for 1 min. The resulting diluted library was transferred to a 2 mL microcentrifuge tube and further diluted with 320 μL Hybridization Buffer to achieve a final loading concentration of 1.8 pM. A 5% PhiX spike-in control was added to the final library.

 

Sequencing

Yeast whole genome sequencing was performed on an Illumina MiniSeq short-read sequencer using a standard Illumina workflow and configured for 2 x 150 bp paired-end reads and MiniSeq flow cell.