Sequencing

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[edit] Introduction

The Huntsman Cancer Institute purchased an Illumina GA II next generation sequencer in early 2008 and a Illumina HiSeq2000 next generation sequencer in 2010. Both of these instruments were placed under the management of the Microarray Core Facility. The GAIIis instrument has been upgrade to a GAIIx. Both instruments are able to support either single read or paired end sequencing with read lengths that range from 36 bases to 101 bases. Bioinformatic assistance with high throughput sequencing data analysis is available through the Bioinformatics Core Facility.


[edit] Illumina Sequencing

The Microarray and Genomic Analysis Core Facility currently has two Illumina HiSeq 2000 instruments and one Illumina MiSeq. Both instruments use Illumina's reversible terminator sequencing by synthesis-based chemistry.

A HiSeq 2000 can deliver 150 to 200 million reads per end (300-400 million reads in a paired end run) in each lane. Standard Run profiles for the HiSeq include 50 bp single read (>85% base calls higher than Q30) and 101 bp paired end (>80% of base calls higher than Q30). We also occasionally run 101 bp single read and 50 bp paired end read sequencing runs. A HiSeq sequencing run takes 3 (50 cycle single end read) to 11 (101 cycle paired end read) days to complete.

An Illumina MiSeq delivers 15-17 million reads per end (30-34 million paired end reads). Standard run profiles on a MiSeq include 25 bp paired end (>90% base calls higher than Q30), 150 bp paired end (>80% base calls higher than Q30) and 250 bp paired end (>70% of base calls higher than Q30). A MiSeq sequecning run takes 6 to 39 hours to complete depending on the run parameters.

[edit] Sample Submission

  • Follow the recommendations below and when in doubt contact Brian Dalley for instructions on sample preparation methods.
  • GNomEx, the Microarray and Genomic Analysis Core Facility LIMS (GNomEx) should be used for documenting experimental details regarding your Illumina sequencing request. All Illumina sequencing requests should be electornically submitted through GNomEx prior to sending samples to the Microarray Core Facility.
  • A GNomEx database account can be setup by completing the information required on the following link ([1]). University of Utah investigators can log in to GNomEx using their uNID and CIS password.
  • Billing for Univesrity of Utah investigators in performed electronically following the completion of work. Prior to initiating an order for Illumina sequencing, the lab group should electoronically submit a work authorization by providing chartfield information for the account to be billed using the following link ([2]).
  • Customers that are not affiliated with the University of Utah can pay either by generating a Purchase Order or by credit card (Visa or Mastercard). A quote for the estimated charges can be provided if needed.
  • GNomEx, the Microarray Core Facility LIMS (GNomEx) should be used for documenting experimental details regarding your Illumina sequencing request. All Illumina Sequencing requests should be electornically submitted through GNomEx prior to sending samples to the Microarray and Genomic Analysis Core Facility. GNomEx will generate database ID numbers for each sample and the database ID number should be inscribed on the top of 1.5 ml microfuge tubes containing your samples. You can inscribe the sample names on the side of each tube.
  • Sequencing access is provided on a first come/first serve basis.

[edit] Sample Preparation and Data Processing

  • Samples submitted for Illumina Sequencing services will be analyzed by appropriate quality control measures (NanoDrop, Qubit, Bioanalyzer, gel electrophoresis) to access the quantity and quality of the sample.
  • Samples that pass quality control steps will be used for generating an Illumina sequencing library using the appropriate sample prep kit. If the quality of a sample is suspect or if there is insufficient sample to construct a sequencing library, a member of teh core facility will contact the investigator.
  • Investigators have the option of constructing the sequencing library within their own laboratory. A fraction (15 ul) of the library can then be submitted to the Microarray and Genomic Analysis Core Facility for sequencing.
  • Following the construction of a sequencing library, an aliquot of the library will be run on an Agilent Bioanalzyer chip to validate the quality and size range of the library. The library will also be qualified by qPCR to establish the molarity of cluster-forming molecules in the library. This step enables us to load an appropriate quantity of the library on a flowcell as a means to optimize cluster density during the sequencing run. We are able to further optimize cluster denisty if additional lanes of the same sample are run on future sequencing runs.
  • Sequencing libraries will then be run on either the Illumina Genome Analyzer IIx or the HiSeq2000.
  • The Illumina HiSeq 2000 and the MiSeq uses real time analysis software in additon to analysis pipeline software to call bases from the image files, generate reads, and deconvolute index tags on the sequencing libraries. Sequence reads will be made available to the investigator for download from GNomEx.
  • Sequence reads can be mapped to a to a genome build of choice by contacting the Bioinformatics Core Facility. See pipeline user guide. Additional bioinformatic analysis can be requested from the Bioinformatic Core. Tools for data analysis that were developed by the Bioinformatics Core may be found at USeq.

[edit] Recommendations for Illumina DNA Sample Preparation

[edit] Recommendation for Illumina TruSeq ChIP Sample preparation

Chromatin immunoprecipitation (ChIP) sequencing enables an investigator to determine the distribution and abundance of DNA-bound protein targets across the genome. We construct ChIP sequencing libraries using the Illumina TruSeq ChIP Sample Preparation Kit [3]. All Illumina TruSeq sample preparation kits enable multiplexing of different libraries within the same sequencing lane. Recomendations are provided below for preparing ChIP samples for Illumina library construction.


NOTE: It is absolutely critical that every ChIP-Seq experiment include a positive control, we recommend H3K4Me3. PolII is also good but doesn't consistently work. Running both would be ideal and a requirement for technicians who are starting out with ChIP-Seq experiments. Barring these controls one cannot troubleshoot issues when they arise nor trust the novel findings. Thus a recommended experiment design is 1 H3K4Me3 chIP, 1 PolII chIP, 3 experiment chIP bio replicas, 3 experiment input control bio replicas. Multiplex all 8 samples in 1 lane single end 50bp HiSeq. ~$2270 for library and seq charges Oct 2013


  • Microcentrifuge Tubes: We recommend the use of Eppendorf LoBind microcentrifuge tubes for all steps of ChIP-seq experiments (Fisher cat# 13-6987-91). LoBind tubes improve recovery by reducing sample to tube binding without the use of any coatings or additives. LoBind tubes are pcr clean and likewise are RNase/DNase-free.
  • Carrier DNA: Do not use salmon sperm DNA, calf thymus DNA or other DNA based carriers as a blocking agent at any step during the immunoprecipitation process. Carrier DNA that is added to your sample can function as template during Illumina library preparation and will contribute to the sequence reads along with your sample.
  • Magnetic Beads: Dynabeads do not readily absorb random DNA from the immunoprecipitation cocktail and therefore are preferred over Sepharose, Sephadex etc.
  • Formaldehyde crosslinking: The concenentration and incubation time of formaldehyde should be optimized by the user. As an initial guideline, a concentration of 1% formaldehyde for 5-15 minutes is common. Use Fisher Scientific catalog #BP531-500 or equivalent as stock source of formaldehyde.
  • DNA shearing and epitope monitoring: Investigators should aim for a mojority of their crosslinked chromatin fragments to be in the 200-600 bp size range while ensuring the integrity of their protein. Fragmentation can be accomplished using either Covaris, Bioruptor, or by tip probe sonicator. This should initially be performed as a time series as a means to optimize the fragmentation time and power settings, monitored by agarose gel electrophoresis. Fragmentation conditions can also be monitored by western blot analysis of aliquots of the input at different time points to monitor the presence of the protein that the ChIP Ab is directed against. Certain proteins are very susceptible to epitope destruction dduring shearing. Determine the time range that is optimal for fragmentation and also optimal for retention of protein.
    • Run an aliquot of de-crosslinked/RNase treated input on a gel and verify that the DNA is in a 100-700 bp size range. Larger sized fragments do not efficiently form clusters on Illumina sequencing flowcells.
    • Define an optimal concentration of cells for shearing and follow that standard in each experiment. Variations in the number of cells used can affect shearing.
    • Tip probe sonnicators may result in variability of size range from one sample to the next. This is the least consistent method of fragmentation, but some investigators find that this method best preserves protein epitopes.
    • When using the Covaris instrument for fragmentation, refer to the most recent protocols available on their website. Keep power level and time length minimized. Be familiar with the current protocols and not the old protocols.
      • Protocol for Covaris truChIP High Cell Chromatin Shearing Kit with non-ionic shearing buffer: [4]
      • Protocol for Covaris truChIP High Cell Chromatin Shearing Kit with SDS shearing buffer: [5]
      • Protocol for Covaris truChIP Low Cell Chromatin Shearing Kit with non-ionic shearing buffer: [6]
      • Protocol for Covaris truChIP Low Cell Chromatin Shearing Kit with SDS shearing buffer: [7]
  • Purification of ChIP and input samples: Samples should be purified using a Qiagen Qiaquick PCR purification kit. ChIP samples can be cleaned up using the Qiagen MinElute PCR Purification Kit which enables elution of ChIP DNA in volumes as small as 10 ul if multiple samples are going to be prepared prior to submitting the ChIP sample to teh core facility. ChIP library construction protocols allow for a maximum of 50 ul of sample per reaction. Avoid the use of organic compounds including phenol/chloroform as these reagents may adversely affect the enzymatic steps invovled in library construction. Likewise, EtOH precipitated samples may contain salts and other contaminants that adversely affect the enzymes used in teh construction of the Illumina sequencing library.
  • Quantification of Sample: The concentration of ChIP samples can be measured by a pico-green assay (the concentration of these samples will be too low to accurately quantitate on a NanoDrop). Invitrogen sells a relatively inexpensive fluorometer, the Qubit, which can be used for this purpose [8]. Quantification of the samples should be done prior to verification of enrichment of known binding sites by qPCR. A minimum of 5-10 ng of ChIP DNA is required for constructing an Illumina sequencing library. If you have less than this quantity, pool multiple ChIP samples together. ChIP samples should be in a volume of 50 ul.
  • Quantification of Enrichment by qPCR: Following the pico-green assay, all ChIP samples should be adjusted to a similar concentration. Create a dilution series of your input DNA that includes the concentration range of your ChIP samples. Perform qPCR using this dilution series of your input sample. Qualify the concentration/enrichment of a few known targets in comparison to a set of negative targets in your ChIP sample and compare these to the concentrations in the input sample. You will need to do a titration series and the two samples being compared should be in a similar concentration range.
  • Quantify the DNA fragment size following ChIP: Run the ChIP samples on a Bioananlzyer DNA High Sensitivity chip and define the size range (samples should be purified in EB using a Qiagen QIAquick PCR purification kit or a Qiagen QIAquick MinElute PCR purification kit). We commonly observe that the average size range of ChIP samples is somewhat higher than the average size range of the input sample that they were derived from. If the size is above 800 bp, the sample can be further fragmented in a volume of 55 ul on the Covaris S2 instrument such that the size range is apprpriate for construction of an Illumina sequencing library (average size of 350 bp). Alternatively, you can attempt further fragmentation using the Bioruptor.
  • ChIP-seq Library Prep: A minimum of 5-10 ng of ChIP DNA is required for constructing an Illumina sequencing library. If you have less than this quantity, pool multiple ChIP samples together. ChIP samples should be in a volume of 50 ul. To ensure the best enrichment ratios, it is important to have an efficient library prep that minimizes PCR cycles, while still yielding enough product for the sequencing step. Library prep involves end repair, adenylation of DNA ends, ligation of adapters and pcr amplification. The size selection step of library construction is avoided by verifying that all ChIP DNA is in an apprpriate size range (200-700 bp) prior to construction of the library. Ten cycles of PCR are performed to enrich the ChIP library. This results in a minimal quantity of DNA to use for sequencing.
  • Validation of enrichment following library construction: If desried, an aliquot of the library can be acquired from the core facility following library construction and before sequencing. Enrichment of known targets and off targets can be validated by qPCR by comparing the ChIP libraries to the input library. All libraries should be normalized to a similar concentration prior to assembling the qPCR reactions. If qPCR fails to show enrichment, you can consider terminating the sequencing request. If ChIP libraries show enrichment, then you can opt to proceed with sequencing.
  • Number of sequence reads: For higher eukaryotes, 10-20 million mapped reads per sample are recommended for performing a whole genome ChIP analysis.
  • ChIP User Experience Link.

[edit] Recommendations for Illumina TruSeq RNA Sample Preparation

[edit] Recommendations for Illumina TruSeq Stranded mRNA Sample Preparation

[edit] Recommendations for Illumina TruSeq Stranded Total RNA Sample Preparation

[edit] Small RNA Sequencing Recommendations

[edit] Exome Capture/Target Enrichment Sequencing Recommendations

[edit] Custom Library Preparation and Sequencing Recommendations

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