NGS submission

STEP 1: Preparation

1. Create a folder in Databases/NGS projects/STS/<date>.
2. Place (or combine) all Sample tracking sheets there. Make sure all barcodes are distinct.
3. Create and fill out a new entry in Lists/Sequencing projects.
4. Pool all samples in a single 1.5 mL Lo-Bind tube.
5. Measure concentration with Qubit.
6. Dilute a small amount of the pooled sample to 1 ng/uL and run Bioanalyzer (protocol).
7. Save the Bionanalyzer report sheet (PDF).
8. Proceed to sample submission. We typically sequence at EMBL GeneCore (detailed below) or (sometimes) in-house.

STEP 2: Choose Illumina Kit

Choices:

• Kit: MiSeq or NextSeq. Depends on what you need based on the choices below.
• Single or paired-end reads: We always choose paired-end
• Read length: Should cover you sample. If paired-end, coverage doubles, e.g., 2 x 150 nt gives covers up to 300 nt.
• Number of reads: We typically aim for 5M reads per sample.

MiSeq

Read Size	5M	25M	50M	100M
1 × 100 bp		25M	50M	100M
2 × 150 bp	10M	50M	100M	200M
2 × 300 bp	10M	50M	100M
2 × 500 bp		50M

Source

NextSeq

Read Size	P1	P2	P3	P4
1 × 50 bp				1.8B
2 × 50 bp	200M	800M	2.4B	3.6B
2 × 100 bp		800M	2.4B	3.6B
2 × 150 bp	200M	800M	2.4B	3.6B
2 × 300 bp	200M	800M

Source

STEP 3: EMBL GeneCore submission

We submit fully prepared libraries, so that GeneCore only has to run them.

1: Fill out the submission form

Open EMBL Genomics Core Facility - Sample Submission

1. User Information: Listed in our internal Laboratory Personnel Onboarding Protocol
2. Sequencer Settings:
   1. Illumina
   2. Illumina sequencing modes: Choose the kit from the table above
   3. Number of sequencing runs: 1
   4. Custom primer: No
   5. PhiX spike-in: Yes, 10%
3. Common Sample Information:
   1. User prepared library: Yes
   2. Pool Concentration (ng/uL): Concentration of the pooled sample as measured by Qubit.
   3. Pool Name / Tube Label: Jones Lab, ==date==. Write the same exact name on the pooled sample tube your submitting.
   4. Application: AmpliSeq
   5. Average fragment size:
      1. Look up the peak sizes (nt) and concentrations (pg/uL) in the Bioanalyzer electropherogram that correspond to your samples.
      2. Compute the weighted average of peaks sizes = sum of peak(i) x concentration(i) / sum of all concentrations.
4. Multiplexing Information:
   1. Number of samples (or samples in the pool): Total number of samples you put in the pooled sample.
   2. Should we demultiplex the samples? Yes
   3. What type of index: Single index
   4. Barcode Length: 8
   5. UMI: No
5. Individual Sample Information: Paste names and indices from the Sample Tracking Sheet (STS).
6. Post-Processing Information:
   1. Alignment required: No
   2. Remarks: If necessary, any other info the GeneCore might need to know, e.g., different length of barcodes
   3. Have you performed QC of the pool via Bioanalyzer or Tapestation? Yes

Download the completed submission file.

2: Deliver samples

1. Place the sample pool in a 1.5 mL Lo-Bind tube with an appropriate label (matching the one in the form) and tightly wrap in parafilm to avoid any leaking.
2. Email the designated sample drop-off person (see the Onboarding document), informing them you’re about to bring the sample and attaching the filled out and signed Packing List that DHL requires. They will contact a DHL courier to come pick up the package from their office. (Typically they show up very quickly, so make sure you have your sample ready.)
3. Deliver the tube to the person's office. They’ll take care of the rest and a few days later will send you an invoice.
4. DHL will send you a tracking number for the package when they pick it up.

3: Submit the submission form

Once you have the DHL tracking number, write an email to solexateam@embl.de (notice that the DHL tracking number must be included in the email, other information is optional):

Hello,

We are from ==XXXX== group at Vilnius University EMBL Partnership Institute and would like to submit our samples for sequencing. We have barcoded the samples ourselves, there are a total of ==XX== samples that are premixed. We have combined 2 ng of our samples, and we have mixed them all in a lo-bind tube, which we hope should reach you within a week (DHL tracking number: ==XXXXXXXXXX==). The concentration of the sample pool was measured with Qubit (==XX.X== ng/uL). Bioanalyzer traces of the final sample pool and the submission form are attached in this email.

If there are any further questions or information needed, please let us know!

Best regards,

==XOXOXO==

Attach:

• Filled-out submission form (from Step 1)
• Bioanalyzer report sheet (from Step 3.1)