Topics of Interest and Frequently Asked Questions
- Starting a project and submitting samples to the ECGC Next Generation Sequencing lab
- Library Prep and Sequencing
- Data Analysis
- Pricing and other frequently asked questions
Do I have to be a Sidney Kimmel Comprehensive Cancer Center Member to use the ECGC services?
No. The ECGC works with researchers from any department or discipline within Johns Hopkins University and beyond. Researchers from outside Hopkins are also welcome to utilize the services used by the core. We have completed projects for researchers from University of Maryland and the USDA, just to name a few. Researchers from outside the institution who receive federal funding for their research will not be charged overhead fees.
How do I start a project with ECGC?
The first step to any project is to set up a consultation meeting, usually involving direct participation of our directors. During this meeting, we will discuss your research project and help you select the most appropriate approach. You do not need to have samples ready for this meeting. The primary focus of this meeting is to learn about your potential project, tailor our approach, and plan analysis to fit your needs. To request a consultation meeting with our directors, please send an email to: ECGC@jhu.edu.
How much DNA/RNA do I need and at what concentration?
Exome-seq: 1ug in 50ul total volume. Lower input protocols are also available.
WGS: 1ug in 50ul total volume. Lower input protocols are also available.
ChIP-seq: 50uL total volume; If you wish to normalize Input amounts to IP samples you must do this before submitting samples, otherwise Input samples will be adjusted to 5ng/uL (250ng total).
RNA-seq: 250 ng Total RNA at a concentration ≥30ng/uL. Lower inputs can also be accommodated for inputs down to 500 pg Total RNA.
miRNAseq: 500ng – 1ug of Total RNA in a minimum of 10ul
MBD-seq: 500ng-1ug in a minimum of 10ul. Lower input protocols also available.
How should I check the concentration? how precise do I have to be?
You can use NanoDrop or Qubit to check the concentrations. Please be as accurate as possible.
What buffers are acceptable?
Water, low EDTA, low salts, Tris buffer.
What kind of tube should I bring?
1.5mL eppendorf tube. NO STRIP TUBES
How should I label my tube(s)
Your name, sample name, date should be written (or via label maker) legibly on your tube. The sample name that you put on your tube should also match the name that you will enter on your sample submission form in iLab, under Tube ID.
What should I name my sample?
The name that you put on the tube must be the same as the name in the sample submission form. You may use any combination of uppercase letters, lowercase letters, and numbers, but please do not use spaces, underscores, or any punctuation.
What kind of QC do you do?
For RNA samples: bioanalyzer and nanodrop
DNA samples: Nanodrop or Qubit
After library prep, bioanalyzer and qPCR are used to QC libraries prior to sequencing.
What happens if my sample fails QC?
We don't want to spend time or money on a sample that is not likely to yield good data. We will inform you immediately if your sample quality is low, and we will not proceed with library preparation until the issue is resolved.
When can I drop off my samples?
Samples can be dropped off at our lab, CRB II Room 131, on Tuesdays and Thursdays between the hours of 10am to 11am and 1pm to 2pm. Please notify us before you plan to drop off your samples. It is important that our staff is present to ensure your samples are handled properly.
What is paired end sequencing?
We can obtain sequence from only one end or from both ends of each DNA fragment in the sequencing library. When both ends of a single DNA fragment are sequenced, this is termed paired end sequencing. The two sequences corresponding to each end of each DNA fragment may or may not overlap with each other, depending on the size of the fragment and length of the sequences. Paired end chemistry is typically used for mRNA, whole genome, and whole exome sequencing. For ChIP and miRNA sequencing, it is often sufficient to have sequence from only one end of the DNA fragment.
How long are the sequences?
100bp, or 50bp. Paired end chemistry is typically 100bp x 100bp (100bp from each end, meaning two reads per DNA fragment). 150 bp sequencing read lengths are also available.
How much time does sequencing take?
The actual sequencing run takes 2-14 days, depending on the instrument and the chemistry. The time needed for sequencing is usually not the limiting factor in a sequencing project. Most projects are complete (including analysis) in 10-12 weeks.
What technologies are available through NGSC?
Illumina Hi Seq 2000
Illumina HiSeq 2500 with Rapid Run capabilities
Illumina MiSeq Personal Sequencer
Agilent 2100 Bioanalyzer
What programs do you use for alignment?
Exome - bowtie2
WGS - bowtie2
ChIP - bowtie
RNAseq - tophat2, rsem
miRNAseq - BLAST, DESeq
MBDseq - bioscope, bowtie2
How many reads do I need?
For some applications, we offer standard "packages" that includes sample QC, library prep, library QC, sequencing, and some analysis. For these packages, we try to achieve the appropriate amount of reads needed for robust biological interpretation according to community and our own in house standards. The target number of reads and the minimum acceptable number for such standard applications is:
Human Exome-seq: Target: 50 million 100x100; Minimum: 10 million 100x100.
Human/mouse/rat low pass whole genome (8-12x average coverage): Target: 150 million 100x100 reads; Minimum: varies.
Human/mouse RNA-seq: Target 50 million 100x100; Minimum: 10 million 100x100.
Human/mouse ChIP-seq: Target 25-50 million 50 bp; Minimum: 10 million 50 bp.
Human/mouse miRNA-seq: Target: 10 million 50 bp; Minimum: 1 million 50 bp.
Human/mouse MBD-seq: Target: 50 million 50 bp; Minimum: 10 million 50 bp.
What is the final output?
Bam and SAM alignment files for all project types.
Exome-seq: variation (when applicable, somatic mutation) calls, SNP calls, Indels
WGS: variation calls, including SNPs, indels, structural variation, and CNVs; somatic mutation calls when applicable; and functional annotation.
MBD-seq: peaks, differential methylation, and functional notation
ChIP-seq: peaks, differential binding, and functional annotation
RNA-seq: spliced alignments, expression tables for both gene and isoform level, differential gene and isoform expression, fusion transcripts
miRNA: counts, differential expression
Can I get additional analyses?
Yes. We can carry out a range of custom analytical approaches as a custom project. Examples include comparing RNA-seq data with methylation or ChIP-seq data, to look for differential expression or isoform usage, or comparing sequence variation with functional data. We have done many more custom analyses, some involving data that we did not generate. Please contact us if you want to discuss an analysis project.
How much does XXX cost?
All pricing can be found on our pricing page.
How many replicates do I need to do?
As many as possible. Sequencing is not immune to biological variability. We recommend at least three replicates; that said, we understand the constraints of budgets and sample procurement and are happy to discuss feasible experimental designs.
Can I get the sequencing done faster?
No. Everybody gets the same standard of service. Because of the way we configure slides and manage our workflow, have no way to guarantee faster completion of a project. Also, sequencing instruments and servers break, reagents go bad, and programs crash, making guarantees impossible. We always do our best to complete projects as quickly as possible.
Can I get a discount?
No. Everybody gets the same standard of service. We can work with you to design an experiment and workflow that fits your budget, but we are not allowed to give discounts preferentially or arbitrarily.
Can you run a different alignment and analysis pipeline on my data?
Yes, we are happy to discuss custom analyses, and these will be priced as standalone analysis projects.
How do I get my data?
We will give you the raw reads, aligned data, and analysis files on a hard drive. Please take care of this hard drive as it is your primary data. We do not keep data on our servers for more than 6 months.