How to submit samples

How to submit your samples

• Minimum 15ul or more
• Minimum 10nM concentration
• Please submit your samples in 1.5-2.0ml tubes
• List of your samples by name on tube
• Index number per sample
• Source ID (Mouse ear tag #, cell line, etc.- something that ties this library back to its source) 
• Sequence length and type (50SR, 100SR, 100PE)
• Bring any custom primers if applicable

Please keep in mind that your samples will be handled often. In order to keep them from getting mixed up, please submit samples in tubes that are clearly labeled and intact (no unattached caps). 

FAQ What Does It Cost

FAQ - What Does It Cost?

The FGC and NGSC cores each have a price sheet posted under the 'Pricing' option in the getting started menu.

The FGC only serves clients in the IDOM/DRC, expect for microarrays which are
open to UPenn and other academic institutions.

In both cases, academics institutions outside of Penn pay a higher amount to make up for the lack of grant overhead.

For maximum flexibility there are separate charges for 

1. sample and library quantification 
2. library preparation
3. sequencing, charged by the lane
4. standard analyses, charged by the sample
5. advanced analyses, only from the FGC.

The exact cost of an experiment depends on many factors, so it is best estimated with the help of the FGC or NGSC technical director after submitting an experiment request.

FAQ Old TessLA Browser

FAQ Old TessLA Browser

Never fear, it's here.

We also have a button on the front page and a link in the Results menu at the top of the page.

Users should begin to use only the portals that are named for the PI, e.g., Eric-Thepi-Lab.

FAQ Where Is My Old Data

1. The cores keep tape back ups of every run.
2. Most runs are kept on our sequencing server for a few weeks.
3. The fastq files and subsequent analyses are stored on the PGFI cluster.
1. The website can be used to download any of these files.
2. Except for very old microarray data that we have archived almost all data is available this way.
3. People with PGFI accounts can access these files directly.
4. Many analyses are attached to tracks in the browser and can be downloaded using our genome browser.

What the Future Holds

The volume of data is quite large and is now a major part of our operating expenses.  Therefore in the future we will be moving to a more aggressive purging of on-line data storage.  We will advise you as plans move forward for this.

FAQ Downloading Data

See this entry.

We have also added a big Data Download button on the home page to take you to your (PI's) download area.

FAQ How Are My Samples Doing?

We will be extending the website and our notification system to provide more information but for now, here's what to expect.

1. Sample Quality Checks
1. takes a few days
2. core staff will usually send an email with the results
2. Scheduling a Run
1. you may have to wait for a flow cell to be full to sequenced
2. this time can vary widely depending on the type of run and our workload.
3. Sequencing
1. once the flow cell is scheduled here are the approximate times
1. clustering - 0.5 days
2. sequencing
1. 50SR - 2 days
2. 100SR - 4 days
3. 50PE - 4 days
4. 100PE - 11 to 14 days
4. Fastq generation
1. usually done the week day the run finishes
5. Basic Analysis
1. ChIP-Seq or HITS-CLIP alignments and peak calling
1. takes 1 to 2 days
2. RNA-Seq
1. take about a week

FAQ When Can I Bring My Samples In?

1. Do not bring samples in until we have told you that the investigation is ready for samples.
2. We accept samples Tue-Thurs from 11-12 and 3-4. On Mondays we only accept samples from 3-4 and on Fridays we only accept samples from 11-12. Also, check the Lab Calendar (found on our website) before coming in to see if there are any events scheduled which would prevent us from accepting samples.
3. If you already have an investigation and want to extend it to include new conditions or assays, then submit a new experiment request.  We will process the request the next morning and once you hear from us you can bring the samples in.

FAQ Does the Core Make Libraries?

FAQ Does the Core Make Libraries?

We offer library preparation services for:

1. RNA-Seq using
1. Illumina truSeq kits 200ng+ of total RNA
2. 
   
2. Agilent SureSelect Exome Capture

We can train you to make other libraries, but do not offer these services.

Why You Should Make Your Own Libraries

We are planning on purchasing a robot to automate this process, but for now library prep is a time-consuming task that can take a while for us to complete.

FAQ How to Make Libraries

FAQ How to Make Libraries

Wow that's a big question!  It's too much to handle in one FAQ, so here are links to individual pages for various library types.

FAQ Getting Started

FAQ - How do I Get Started?

The basic things you need to do are:

1. Read the other FAQs about experiment design and library prep
2. If you still have questions, check the Consultation Calendar and propose a time to meet to discuss your questions.
3. Have your PI create an account (this only needs to be done once.)
4. Create an account for yourself (this only needs to be done once.)
5. Submit an experiment request.
6. The next morning we will review experiment requests and contact you to resolve any questions.
7. We will notify you that we can accept samples.
8. Bring samples by at either 11-12 or 2-3 Monday to Friday.

FAQ Which Core To Use

There are a few Cores at Penn that do DNA sequencing.  Here is what we offer

NGSC

The NGSC has 3 Illumina hiSeq2000s and a miSeq. Here is what these machines are good for:

hiSeq2000

The hiSeq2000 is good for these techniques (and their many variations) RNA-Seq, ChIP-Seq, miR-Seq, HITS-CLIP, exome capture, BIS-Seq, and whole genome sequencing in mammals.

There are two aspects of ultra-high throughput sequencing that are important counts and coverage.  Counts are important for RNA-Seq, ChIP-Seq, miR-Seq, and HITS-CLIP.  Coverage is important for exome capture, BIS-Seq, and whole genome sequencing.   The hiSeq2000 generates sequence for about 200 million fragments per lane.  For each fragment the hiSeq can produce single or paired-end 50bp or 100bp sequences.  Using 100bp pair-end sequencing, you can get up to 40Gb per lane.

In many cases a single lane can generate more counts or coverage than a sample needs.  In that case, it is important to use multiplexed adapters so we can sequence multiple samples per lane.  Multiplexed adapters are generally a good idea is they allow samples to be test sequenced for quality, then sequenced deeper as needed.

Technique	Typical Volume	Samples per Lane
RNA-Seq	30 to 200 million reads	1 to 6
ChIP-Seq	30 to 100 million reads	2 to 6
miR-Seq	10 million reads	20
HITS-CLIP	30 million reads	6
Exome capture	20-30x coverage	5 to 20
BIS-Seq	20-30x coverage	1/3
Genome Sequencing	20-30x coverage	1/3

miSeq

The miSeq uses the same libraries as the hiSeq2000. It generates only about 15 million fragments per lane, but runs very quickly, and can generate reads as long as 150bp (or longer).

It is good for sample testing, miR-Seq, amplicon sequencing, or the techniques above applied to small, e.g., bacterial genomes.

FGC or NGSC - which core to use?

The Functional Genomics Core (FGC) and the Next-Generation Sequencing Core (NGSC) provide similar services, but with some important differences.
Here is a summary to help you decide which core is right for your project.

FGC

• high-throughput sequencing for IDOM/DRC members
• downstream data analysis for IDOM/DRC members as capacity allows
• Agilent microarrays for IDOM, UPenn, and academic clients
• limited RNA-Seq library prep for IDOM/DRC members

NGSC

• high-throughput sequencing for UPenn and academic clients
• standardized basic preliminary data analysis for UPenn and academic clients
• limited RNA-Seq library prep for UPenn, and academic clients

For the NGSC and FGC, prices are higher for external clients.

To good news is that you talk to the same people no matter which core you use.

FAQ-GettingStarted

Getting Started

Here is what you need to do to get started with the core.

1. Lab PI makes an account.
2. Experiment investigators make accounts under the PI.
3. The next weekday morning the core staff activate the accounts.
4. Create a New Experiment.
5. The Core staff read your description then either load the experiment or contact you to ask for clarification.
6. Once the core has loaded the investigation, you can bring your samples.
7. We do quality checks on your samples and let you know how they look.
8. We sequence the samples.
9. We do the data analysis you requested.

 For subsequent experiments, pick up at step 4.

How the Core Works

Overview

Here are major steps involved in doing an experiment with the NGSC.

1. Create accounts at the NGSC or FGC website using the Create New Account link.
• The PI of the lab should create the first account.
• The investigator(s) who will actually do the experiment should then create their account(s).
• Creating a PI's account works best on Safari on a mac - we are fixing this!
• Investigators should make sure they pick their PI from the list.
• We will active the accounts within a day or so.
2. If necessary meet with the technical director to help design the experiment.
• Use the Appointment Calendar link to identify a time to meet.
• Send him an email to propose the time.
3. Create an experiment use the Create New Experiment form.
• This is a basic description of the experiment you want to do.
• The web page indicates the info we are looking for.
• Please include your billing information.
4. We will formalize the experiment description and load it into the database.
• This process takes a few days.
• We may send emails to clarify details of the experiment.
5. Once we have loaded the experiment, bring your samples to the core.
• Please do not bring the samples until the experiment has been loaded.
• Be prepared to provide a source ID and a sample name for each sample
6. We will check the quality of the samples and report any problems.
7. We will schedule the libraries for sequencing and report our progress.
8. Data analysis will follow sequencing as soon as possible.

On-Line Tools

Here is a short list of on-line tools for genome data analysis and/or visualization:

• DAVID/EASE
• functional analysis
• http://david.abcc.ncifcrf.gov/
• HOMER
• ChIP-Seq and other data analysis
• http://biowhat.ucsd.edu/homer/ngs/index.html
• CISTROME
• ChIP-Seq analysis
• http://cistrome.org/Cistrome/Cistrome_Project.html
• GALAXY
• ChIP-Seq and other analysis and visualization
• https://main.g2.bx.psu.edu/
• UCSC Genome Browser
• http://genome.ucsc.edu/

Downloading Data

The Basics

We have configured the website to use your account usernames and passwords to access all files by PI (if you are a PI or lab member) or by investigation if you have collaborator status.

We have updated the download area to provide all data at a URL link this:

  https://fgc.genomics.upenn.edu/Experiments/PI

You will be prompted for a user name and password, enter the credentials you use to log in to the rest of the site.

If you don't find the data you are looking for, check the deprecated links desribed below and let us know of the omission.

How the Files are Organized

Under each PI are a number of folders that correspond to experiments (the old style) or investigations and (under that) studies (which is the new style.)

Within each experiment or investigation, there are few common places to look for data.

'Raw' Data

This data is not really raw, but has not undergone any real analysis beyond alignment

• basic/Fastq - files of read sequence in FASTQ format
• basic/Fasta - files of read sequence in FASTA format
• basic/Solexa - older style sequence or ELAND output files
• basic/Export - alignment information split into unique and not-usable (repeat) 
• basic/BedFiles - BED file format of uniquely aligning reads and perhaps SHP output files

Analysis Results

These are typically places under the Analysis folder.  Different types of analysis are organized in subfolder under that.

Tips and Tricks

WIG files for Profiles

At the moment, we generate profile data at full resolution, i.e., including the changes resulting from each read.   Sometimes, these files can be found in basic/BedFiles with names in the form FGCNNNN_s_L-ucsc.ushp.tab.gz, e.g., FGC0138_s_7-ucsc.ushp.tab.gz.  These files are not in WIG format - they are just chromosome, begin, end, score.  They can be converted to WIG format with relatively simple programs.

WIG format data can be obtained using the download feature in the TessLA browser.  However, because modern data sets are so large this approach is not very effective.

Depcreated URLs

Because the files sit on two different filesystems you have to look at two, slightly, different URLs:

  https://fgc.genomics.upenn.edu/Experiments-1/PI

or


  https://fgc.genomics.upenn.edu/Experiments-2/PI

where, of course, you replace PI with your PI's last name.  Note the https, that's important.

Scheduling Runs

Policy

Since hiSeq2000 flow cells contain 8 lanes, we need to collect 8 lanes-worth of libraries before we begin a run.  This is more than most investigators will need to get an experiment started and, often, more than they will have prepared at a time, especially with multiplexing.  Therefore we take care will schedule flow cells from libraries from the sequencing queue in a first-come first-served fashion.

Because we do this scheduling it is actually counter-productive for clients to fill a flow cell and insist that the samples be sequenced together. There is rarely a technical reason to have everything sequenced on the same flow cell, so we ask that you let us schedule the flow cells to get maximum throughput.

Sequencing Queue

We have sequencing queues for each combination of sequencing parameters:

• length (50 or 100bp)
• paired-end or single-read
• multiplexed or plain

At the moment 50bp PE sequencing is rare, so this is the one case where bringing 8 lanes of libraries would be helpful.

If you can't wait for a 50bp PE flowcell to fill, then we will put the library in the 100bp PE queue.

How to Find Us

We are located in the Translational Research Center on the 12th floor.

12-156 Translational Research Center

3400 Civic Center Blvd Bldg 421

Philadelphia, PA 19104-5156

Floor Plan