Next Generation Sequencing Core

All sample drop-offs must be accompanied by the appropriate submission forms.

1. Please download appropriate submission forms below and fill out completely according to instructions on the first sheet.
   • Illumina LIBRARY & SEQUENCING FORM: RNA and DNA sample for Illumina library prep and sequencing
   • Illumina SEQUENCING ONLY FORM: Submit your library for HiSeq or NextSeq sequencing
   • PacBio_Sequel II LIBRARY & SEQUENCING FORM
   • Nanostring nCounter Form
2. Email completed Sample Forms to SampleSubmission@Salk.edu.
3. Sample Drop off time: Tuesday and Thursday 10:00 a.m. – 12:00 p.m. (Holiday excluded)

1. RNA /DNA Sample Requirements for Illumina Library Preps
   • The input DNA and RNA are quantified by a fluorometric method (e.g. Qubit, PicoGreen).
   • Provide nanodrop reading if you do not have access to a fluorometric method .
   • All RNA samples have to be DNA free/All DNA samples have to be RNA-free.
     • Illumina LIBRARY & SEQUENCING FORM: RNA and DNA samples for Illumina library prep and sequencing
     • Illumina SEQUENCING ONLY FORM: Submit user-prepared libraries Illumina sequencing

 

Type of Library	Concentration (determined by Qubit)	Total Input Amount	Quality	Comments
Stranded mRNA (PolyA-selected)	Total RNA 50-200 ng/µl	>1 µg	RIN > 8 A260/280=~2.0 A260/230=2.0-2.2	Resuspend in RNase-free water
Total RNA (Ribosomal & Mitochondrial RNA depleted)	Total RNA 50-200 ng/µl	>1 µg	RIN > 8 A260/280=~2.0 A260/230=2.0-2.2	Resuspend in RNase-free water
Ultra Low Input RNA	0.1-10 ng		RIN > 8 A260/280=~2.0 A260/230=2.0-2.2	Resuspend in RNase-free water
Whole Genome (Illumina DNA PCR free)	20-500 /uL	>500 ng	A260/280=1.8-2.0 A260/230=2.0-2.2	Resuspend in EB (Tris-HCl 10 mM, pH 8.0)
Small Genome (>5Mb), Amplicon (>150bp) (Illumina DNA prep old name Nextera flex)	5-50 ng/uL	>100 ng	A260/280=1.8-2.0 A260/230=2.0-2.2	Resuspend in EB (Tris-HCl 10 mM, pH 8.0)
Small Genome (<5 Mb), Amplicon (>300 bp) (Nextera XT)	0.2-1 ng/uL	>2 ng	A260/280=1.8-2.0 A260/230=2.0-2.2	Resuspend in EB (Tris-HCl 10 mM, pH 8.0)
ChIP, Fragmented DNA	0.2-5 ng/uL	10 ng	A260/280=1.8-2.0 A260/230=2.0-2.2	Resuspend in EB (Tris-HCl 10 mM, pH 8.0)

 

2. User Prepared Library Submission and Requirements for Illumina Sequencing
   Pooled Libraries should be quantified by a fluorometric method (e.g. Qubit, PicoGreen) and submitted with TapeStation traces (or equivalent). Please submit your pooled libraries in clearly labeled 1.5 mL microcentrifuge tubes (low-binding preferred). We quantify all library pools before loading them onto the sequencers.

 

User Prepared Library	Concentration (determined by Qubit)	Volume	Preferred Buffer
NextSeq, MiniSeq	5 nM (Approx. 0.5~1.0ng/µl)	30 µl	EB (Tris-HCl 10 mM, pH 8.0) or EB+0.1%Tween20
NovaSeq S4 Flowcell	5 nM (Approx. 0.5~1.0ng/µl)	300 µl	EB (Tris-HCl 10 mM, pH 8.0) or EB+0.1%Tween20
NovaSeq SP, S1, S2 Flow cell	5 nM (Approx. 0.5~1.0ng/µl)	150 µl	EB (Tris-HCl 10 mM, pH 8.0)

Sample Submission Requirements for SMRT bell Template Prep
For HMW gDNA, DNA quality will affect the resulting library and sequencing results. It is imperative that the DNA used is at high quality and sufficient quantity since there is no amplification step.
 

Application	Input	Total Input	Concentration (determined by Qubit)	QC	Comments
ISO-Seq (full length transcript RNA-Seq)	Total RNA	>700 ng	50-200 ng/µl	A260/280=1.8-2.0 A260/230=2.0-2.2 RIN > 8	Resuspend in RNase-free water
	cDNA	>1 µg	50-200 ng/µl	cDNA Profile (Tapestaion or Bioanalyzer)	Resuspend in EB or low EDTA TE
WGS, ~10kb, CLR	HMW gDNA	>5 µg	50-200 ng/µl	A260/280=1.8-2.0 A260/230=2.0-2.2 PFGE images for gDNA	Resuspend in EB or low EDTA TE **DNA should be at high quality and sufficient quantity since there is no amplification step.
WGS, >15 kb, CLR		>15 µg	50-200 ng/µl
WGS, >30 kb, CLR		>25 µg	25-200 ng/µl
High Fidelity (HiFi), 10-15 kb, CCS		>15 µg	100-500 ng/µl
Purified PCR products/ Multiplexed amplicons	Amplicon Size	Input DNA Mass per Amplicon Pool	Required Conc. per Amplicon Pool	Size Distribution (Tapestaion or Bioanalyzer)	Resuspend in EB or low EDTA TE
	250-500 bp	1 ug	15-100 ng/µl
	500-1000 bp	1 ug	15-100 ng/µl
	1-3 kb	2 ug	30-200 ng/µl
	3-10 kb	4 ug	50-400 ng/µl

The listed equipment overseen by NGS core are available for the Salk community. 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. All labs using facility equipment must have their personnel trained. This will help us monitor machine use and ensure that everyone has the same knowledge base. Additionally, please make sure to clean after your use for the next users and return any core equipment where they belong.  

• 10X Genomics Single Cell Solution (Requires Reservation)
• Q3 and Q5 QuantStudio RT-qPCR system (Require Reservation)
• Agilant Bioanalyzer 2100
• Agilant TapeStation 2200
• Qubit Fluorometer 2.0
• Covaris™ M220 Focused-ultrasonicator™
• EpiShear™ Probe Sonicator
• Bio-Rad QX200 Droplet Digital PCR System
• TECAN Infinite^® 200 PRO reader
• 3D Printer (Require Reservation)

Instrument Reservation https://secure8.ideaelan.com/secure/public/applogin.aspx

• You can log in with your AD account credential.
• Select “Schedule Instruments”
• Select Facility (upper right corner) → Next Generation Sequencing
• Please read reservation rules in Infinity

Service Guide

1. Inoculate colonies in up to 5 ml LB or TB with appropriate antibiotic in 14 ml Falcon tubes (BD 352059). Close cap loosely. Label the tubes with first and last initial followed by a numerical ID (Example AB01, AB02, etc.) Place samples in the first numerically available slot in the 37 degree shaker on the fourth floor of the south building, directly above NGS core. The samples will be processed in the numerical order on the slots.
   Note: 14 ml Falcon tubes are the only tubes that will be accepted by the Robot. Samples sent in all other kinds of tubes will not be processed.
2. Fill out the spreadsheet on the top of the shaker. Make sure the slot ID matches the spreadsheet ID.
3. To minimize the handling error and for easy distribution of the DNA, the number of samples submitted should be in multiples of 8. For example: 8, 16, 24, etc. The minimum number of samples is 8. You will be charged for the cost of 8 samples even if you submit only 6 samples.
4. Samples will be done before noon the following day.
   Note: In order for your samples to be processed the following day, you have to grow your cultures using core’s designated shaker.
5. You will be notified by email when your samples are ready to be picked up. Please pick up your samples by 3:00 pm that day.
   Note: The Functional Genomics Core Facility will not be responsible for any samples that are not picked up by this time.
6. For those of you who have 96 samples altogether, the core will provide you with a 96 deep well block for inoculation. This will save you the time and effort of inoculating in tubes.