🔹FAQ: Sample Submission (Last updated: Aug 16, 2021) 

2. Can I drop samples anytime when NGS is open?

Yes.

Our regular sample drop-off time is Tuesday/Thursday between 10 am and 12 pm. Feel free to drop off your samples during this time without prior notice. To make alternative arrangements, e-mail ngs@salk.edu.

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3. How to fill the submission form?

To request sequencing services with or without library prep - after logging into PPMS, navigate to the Integrative Genomics Core (IGC) tab and click “request/start a new project” on the home screen. You will then be prompted to fill out a sequencing request form. Sequencing requests will no longer be accepted by e-mail. However, the Core is always available to answer any questions at ngs@salk.edu.

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6. How to label sample tubes?

• For DNA/RNA samples and individual library (pooling service needed) samples

Please put your samples in individual-cap PCR strips (pic showed on the right) and label your samples with consecutive numbers followed by your initial (which is the same as sample code). 

For example, Michael Jones labels  tubes as MJ1, MJ2,MJ3.......

• For library pool(s)

Please put your pool(s) in 1.5 mL tube and label with your initial  and the same submission date in the form.
For exmaple, MJ-4/20/2021-p1*, MJ-4/20/2021-p2 ...(*p means pool, if there are more than one pool in the submission form)

**Don’t cut your strips; keep it as whole strips.

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7. How to QC my samples?

NGS core has both Qubit and TapeStation 2200 to QC any DNA/RNA samples.

Qubit will give you an accurate reading of your sample concentration, while TapeStation can provide you a RIN/DIN number, indicating the integrity of your samples. Please find the instruction sheet for each instrument in the first drawer under the QC bench. 

All the reagents and tapes are inside the 4˚ fridge under QC bench. Please refer your nanodrop concentration to decide if high sensitivity tapes and qubit reagents are needed. If you are not sure, please make an appointment with one of our staff for overview/training. We are happy to show you how to do QC.

Please reserve the QC bench ahead before planning your trip to NGS Core (reservation calander link).

• NGS Qubit 2.0 instruction PDF download

• NGS Tapestation 2200 instruction PDF download

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8. Can NGS still generate libraries for those samples which are below the QC threshold? 

We can still try to generate libraries for those samples that didn’t meet our QC requirement but can’t guarantee the success of the library generation. You will be responsible for the cost of libraries preparations if they are failed. Please note that low quality RNA and DNA for library prep affect your data quality for analysis.

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10. When can I receive the data?

We can’t give you a firm answer, it depends on sequencing platform you choose and the order in the queue. Usually will be between 2-4 weeks.

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🔹 FAQ: NGS Shared Instruments (Last updated: April 19, 2021) 

11. What instruments are shared in NGS core?

In order to maintain the longevity and proper functioning of all the equipment, please have proper training before use and follow the instruction very carefully. 

• Q3 and Q5 QuantStudio RT-qPCR system (Require Reservation)

• Agilent TapeStation 4150 (Require Reservation)

• Qubit Fluorometer 3.0 (Require Reservation)

• TECAN Infinite® 200 PRO reader (Require Reservation)

• 3D Printer  (Require Reservation)

Please use this link to reserve the date/time. 

https://ppms.us/salk/listsys/?pf=9

**Go to question 12 to see "how to make a reservation"

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12. How to reserve NGS self-service instruments?

Instrument Reservation

Please use this link to reserve the date/time. 

https://ppms.us/salk/listsys/?pf=9

• You can log in with your AD account credential.

• Select “Book”

• Book any instrument you are going to use.

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13. How to enter the recharge for NGS self-service?

The use of qPCR and 10x machine will be charged automatically with the reservation time selected.

All other instruments are charged only with the quantity of consumables/reagents used from NGS. Please enter the quantity of tubes/tapes/reagents into recharge computer.

**Log recharges if you used this servise/ instrument list below

• Tape Station (self Service or machine use only) 

• Qubit Quantitation (self service) 

• Tecan plate (purchase from core)

• Ampure XP beads or SPRI beads 5mL aliqouts (purchase from core)

• 3D Printer Usage 

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🔹 FAQ: Library and Sequencing (Last updated: April 19, 2021)

14. What sequencers does NGS have?

We have 

• short read sequencer--Illumina Miniseq, Nextseq 2000, Novaseq X, Element AVITI.

• long read sequencer--Pacbio Revio, Nanopore GridIon

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15. Sequecing, library making, and other service rates ?

➡ 

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16. Which sequencer platform and run configuration (sequencing read length and reads) should I choose on the form?

It depends on number of samples you have and depth of sequencing you need for each sample. Here is a brief read length recommendation from Illumina website, but keep in mind, sequencing configuration must be changed accordingly by following your experiment design. The information of sequence platform and amount of data reads can be found in a submission form (instruction page) or on Salk NGS core official webpage.

• Illumina Read length recommendation (click the link to get more detail information)

• Considerations for RNA-Seq read length and coverage (click the link to get more detail information)

How many reads should I target per sample?

Gene expression profiling experiments that are looking for a quick snapshot of highly expressed genes may only need 5–25 million reads per sample. (20-30M reads /sample recommended)

How long should my reads be? 

Gene expression / RNA Profiling – Quantifying the coding transcriptome typically requires a short single read (often 50–75 bp) to minimize reading across splice junctions while counting all RNAs in the pool. (Single end 50bp or pair end 50bp recommended)

• Illumina sequencing coverage calculator (click the link to get more detail information)

• Salk NGS Core rates 

You can make an appointment to consult with NGS core director or IGC core members about your experiment design and sequencing configuration. Or you can follow the sequencing suggestion in your library kit protocol (for examples, 10x single cell user guide)

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17. Which library prep method should I choose, mRNA library or Total RNA library?

TruSeq Stranded mRNA specifically targets poly-adenylated RNA (including coding RNA and non-coding with poly adenylation), while TruSeq Stranded Total RNA prepares RNA libraries based on rRNA depletion, targeting coding RNA and long non-coding RNA.

TruSeq Stranded mRNA requires high quality RNA (RIN>8) as input and TruSeq Stranded Total RNA can work with degraded / FFPE samples.

• More FAQ about Illumina RNAseq, please see here. 

**Kits information can be found in Question 18

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18. What kits does NGS Core use for library prep?

We currently offer Stranded mRNA library prep, Total RNA library prep (rRNA depletion), Small RNA library prep, WGS library Prep (PCR free or low PCR cycles), Low Input DNA library Prep (for CHIP), Low Input RNA library prep, 10X 3’scRNA library construction from cDNA, others (special cooperating project approved by NGS director including Pac-Bio and Nonopore sequencing project)

Click each item and link to user guide

Illumina RNA library prep

• Truseq Stranded mRNA library prep

• Truseq Total RNA library prep (rRNA depletion Ribo Gold)

Whole genome sequence library Prep (PCR free or low PCR cycles)-

• Illumina DNA PCR-Free Library Prep, Tagmentation

• Illumina DNA Prep ( same as Nextera flex)

• Nextera XT DNA Library Prep

Low Input DNA library Prep (for CHIP)

NEBNext® Ultra™ II DNA Library Prep Kit for Illumina®

Low Input RNA library prep

• SMART-Seq® HT Ultra® Low Input RNA Kit for Sequencing (with Nextera XT for downstream library construction) (RNA to cDNA)

• Nextera XT DNA Library Prep (cDNA to Illumina library)

10X 3'scRNA library construction from cDNA

• 10X 3’scRNA V3.1 single index library construction from cDNA

PacBio (Sequel IIe) 

• genomic DNA library prep

• Multiplex Structure Variation library prep

• HiFi library prep (High accuracy WGS sequencing)

• Iso-seq (full-length transcript) RNA library prep

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19. My libraries have adaptor dimer contamination, what should I do?

You could either do another round of bead purification or electrophoresis gel purification.

If your library concentrations are low, it is not recommended to do bead purification because of low efficiency. It could work only when you have quite amount of samples.

Gel purification is tricky but most efficient way to remove the adaptor dimers. The recovery rate differs. It is also not recommended when you don't have enough samples.

You could sequenc the samples if the samples are essential and precious and adaptor dimers are less than 1%. It may end up losing ~5% of the reads since the adaptor dimers tend to cluster faster than your libraries. Or do the gel purification to cut off the contaminants with the chance of losing your libraries (you may pool the libraries first, so you have more DNA to start with). 

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20. My pool concentration is too diluted. What should I do?

You can use speedvac to concentrate your pool to meet the desired concentration.

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