Difference between revisions of "Sequencing"

From Genome Technology Core (GTC) wiki - Sequencing and Microarray
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= TECHNOLOGY =
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|title=Sequencing Service
 
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The GTC offers Next-Generation sequencing on the Illumina's NovaSeq and Element Bioscience's AVITI platform. More details on the [[Pricing| pricing]] page.
  
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GTC has two Illumina Genome Analyzers 2.0. Each sequencer can process up to 16 samples per week, with a data yield of 20-30 million reads per sample. Read lengths are 36 to >100 bases in single or paired-end formats.  
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We also have Nanopore PS2 solo for long read sequencing.
  
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Templates are “sequenced” by incorporating fluorescently labeled nucleotides with Illumina's proprietary “Sequencing-By-Synthesis” technology. A detailed description of this process can be found here: http://illumina.com/pages.ilmn?ID=203
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= LIBRARY PREP SERVICE =
  
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= Sample Submission Guidelines =
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We offer many options for library prep for a wide range of applications and input levels down to RNA from single cells. Please refer to the pricing page for more information.
  
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Users are required to prepare their own sequencing libraries. Samples should be gel-purified and provided UNDILUTED in EB.
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= PRICING =
  
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Users should submit 5uL of undiluted sample. We will determine the concentration by RT-PCR and make the appropriate dilutions. Excess sample will be retained in our freezers for potential future re-run. Users are also strongly encouraged to retain a portion of their samples in case of emergency.
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For pricing information - [[Pricing| Click Here]]
  
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GTC will provide sequencing primers for libraries prepared with standard Illumina adapters. If custom sequencing primers are required they must be provided at the time of sample submission.
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{{SeqSubmissionGuide}}
  
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= Data =
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= DATA =
  
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Images acquired from the solexa sequencer are processed through the bundled solexa inage extraction pipeline to get the sequence and quality score for each base. The data is aligned to a reference genome, if requested, using an interative ELAND algorithm. An in-depth QC report is included in the package.
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Images acquired from the sequencer are processed through the bundled Illumina image extraction pipeline to get the sequence and quality score for each base. An in-depth QC report is included in the package.
  
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= Protocols =
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* QC Details - [[SequencingQC|Sequencing]]
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* Formats Details - [[SequencingFormats|Sequencing]]
  
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Please refer to the protocols section here - [[Protocols]]
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== TURNAROUND TIME ==
  
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== Turnaround Time ==
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2-3 weeks
  
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Each Genome Analyzer processes 8 samples per run, or 7 samples plus a control. Full flowcells can be run usually within two weeks of submission. Partial submissions of less than eight samples (or 7 with control) are put into a project queue, where they join existing samples or await others before processing. Wait times for partial submissions vary but are generally two weeks or less, depending on demand from other users.
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= APPLICATIONS =
  
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= Applications =
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Next-Generation Sequencing can be used for a wide variety of applications including ChIP-Seq, RNA-Seq, smallRNA-seq, GWAS and much more. Visit Illumina's web site or contact us to discuss your project goals.
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== ChIP-Seq ==
 
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ChIP-Sequencing (or ChIP-Seq) combines chromatin immunoprecipitation (ChIP) with DNA sequencing allowing researchers to identify the binding sites of DNA-associated proteins.
 
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== Small RNA Sequencing ==
 
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Small RNA discovery provides identification of non-coding RNA to aid in the understanding of gene regulation. It can be helpful in identifying novel small RNA molecules. http://illumina.com/downloads/rnaDGESmallRNA_Datasheet.pdf
 
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== mRNA Sequencing ==
 
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We offer a mRNA Sequencing service utilizing the Genome Analyzer System (Solexa) from Illumina. Primarily, we offer:
 
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* Digital Gene Expression Sequencing Service <br>
 
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Digital Gene Expression is similar to SAGE analysis. Sequencing of short mRNA “tags” provides genome-wide expression profiles and is used as an alternative to full-genome microarray hybridization or qPCR. Digital Gene Expression is more sensitive and has a greater dynamic range than traditional microarrays allowing for more accurate quantification and detection of very low abundance transcripts. http://illumina.com/downloads/rnaDGETagProfiling.pdf
 
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* Whole Transcriptome Analysis <br>
 
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Sequencing the entire transcriptome can reveal valuable information about exon boundaries and splice variants that may be missed by microarray analysis or Digital Gene Expression. Kits for whole transcriptome analysis are expected to be available from Illumina soon.
 
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== Denovo Sequencing ==
 
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De novo sequencing can be done using the Genome Analyzer, although the short read lengths can make assembly challenging, especially in larger genomes. However, advancements are being made to increase read length and improve assembly algorithms. http://illumina.com/downloads/ch2-ILMN_ProdGuide_DNAsequencing.pdf
 

Latest revision as of 16:51, 10 July 2024

TECHNOLOGY

The GTC offers Next-Generation sequencing on the Illumina's NovaSeq and Element Bioscience's AVITI platform. More details on the pricing page.

We also have Nanopore PS2 solo for long read sequencing.

LIBRARY PREP SERVICE

We offer many options for library prep for a wide range of applications and input levels down to RNA from single cells. Please refer to the pricing page for more information.

PRICING

For pricing information - Click Here

SEQUENCING SUBMISSION GUIDELINES

STEP 1: REGISTRATION (FOR NEW CUSTOMERS ONLY)

  • INTERNAL CUSTOMERS: Do NOT require to register in the system

All external customers please obtain a purchase order number prior to your submission. If you need a quote, please email us at wibr-genome@wi.mit.edu or call us at 617-258-8803. Blanket purchase orders are preferred, if you plan to submit multiple projects over time.


STEP 2: PREPARATION OF SAMPLES

SUBMISSION OF SAMPLES FOR LIBRARY PREPARATION

Samples should be in a buffer that does not contain trace amounts of phenol or EDTA. We will save and return any unused input material after libraries are successfully prepared.

Below are our input requirements for Library Prep Service

Sample Type Sonication/Fragmentation* Fragment Size (bp) Input requirements (ng)** Volume (ul)
Genomic Required 200 to 400 500 to 1000 10-50
ChIP Required Variable (specify) 10 to 50 10-40
RNA (total) NA NA 100 to 1000 10-50

*Unless special arrangements are made with us in advance, we will assume that your genomic DNA sample has been fragmented, by sonication or otherwise, prior to submission.

**We can work with less if necessary, but it is best if you elute your DNA in 50 ul or less if you believe we may need to use all of it. Please give us at least 10uL of sample to ensure we have enough for QC and prep.

SUBMISSION OF PRE-PREPARED LIBRARIES

We typically need 10-20 ul of each library at 5 ng/ul or greater. If your library is less concentrated than 5 ng/ul, we’ll quantify it to make sure we have enough to sequence.


STEP 3: ONLINE SUBMISSION OF SEQUENCING LIBRARIES

  • Please complete the online submission form (Link To Form Available Here). Questions or problems related to the submission should be directed to Sumeet Gupta at sgupta@wi.mit.edu
  • Every physical sample tube should have its own Genome Tech Core ID (GTC ID) number (automatically generated and emailed to you immediately after submission of the online form), even if you plan to multiplex them.
  • Please write your name GTC ID numbers on the tubes. We prefer 1.5 ml tubes.


STEP 4: DROP-OFF OR SHIPPING

When you are ready, you may hand deliver or ship samples them on dry ice to:

Whitehead Institute Genome Technology Core
455 Main Street, Room 325
Cambridge, MA 02142

Please do not ship without first contacting someone in the lab, to make sure that we are here to receive your package. Do not rely on voicemail. Please wait until you hear back from someone.

DATA

Images acquired from the sequencer are processed through the bundled Illumina image extraction pipeline to get the sequence and quality score for each base. An in-depth QC report is included in the package.

TURNAROUND TIME

2-3 weeks

APPLICATIONS

Next-Generation Sequencing can be used for a wide variety of applications including ChIP-Seq, RNA-Seq, smallRNA-seq, GWAS and much more. Visit Illumina's web site or contact us to discuss your project goals.

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