Next Generation Sequencing

Next Generation Sequencing is now being offered at our facility using Illumina's MiSeq platform.

 

The MiSeq uses a "Sequencing by Synthesis" concept whereby prepared libraries are loaded onto the MiSeq flow cell, cluster generation is performed and sequencing by synthesis (SBS) occurs.    

Reagent kits available are as follows:

2 x 75 (150 Cycles) v3

2 x 300 (600 Cycles) v3

2 x 25 (50 Cycles) v2

2 x 150 (300 Cycles) v2

2 x 250 (500 Cycles) v2

Plus some nano kits

v3 Chemistry gives approximately 25 million total reads

v2 Chemistry gives approximately 15 million total reads


Dual index sequencing is available with the capacity to multiplex up to 96 samples in one run.  The coverage you require will determine how many samples can be multiplexed and run at one time. 


The Lander/Waterman equation is a method for computing coverage

The general equation is:  C = LN/G

C stands for coverage

G is the haploid genome length

L is the read length

N is the number of reads

For example using a 2 x 300 v3 kit that gets 25 million reads and run a 5.4 Mb bacterial genome

C = (600)(25,000,000)/5,400,000 = 2777 coverage

If you wanted 80X coverage per sample, you could multiplex 34 samples.


Sequencing Libraries you generated:  

Do you want to save money by making your own libraries?  Great!  We can still help you if you have questions and we can run your samples usually within a few days.  

Instrument run cost:  $500

Plus the cost of the sequencing kit including shipping.   


Cost:  Costs vary depending on the number of cycles you need and the type of library prep needed.  Please inquire about pricing for individual projects with Jodie.  

  • We will break down the individual cost of each project for you rather than just giving you a generic library prep cost.
  • As an example, we ran 24 bacterial genomes using a 2 x 300 sequencing kit and Illumina's Nextera Library prep kit at the cost of approximately $188/library.  


We partner with the Genomic Analysis and Sequencing Core Facility located in the SOPH. They have extensive experience with extractions.  

Please visit their website to see a list of their services. 

​Applications include:
Targeted Resequencing
  • Amplicon Sequencing
  • Hybrid Capture
  • Clone Checking
Small Genome Sequencing
  • De novo
  • Resequencing - includes alignment to your reference genome
  • Plasmids
RNA Sequencing
  • 16S Metagenomics
  • Small RNA Sequencing
Quality Control
  • Library QC
Regulation
  • ChIP-Seq usually for QC before scaling up to a HiSeq run
   
 

 

 

The steps to generating sequence include the following:

  • Consultation.  We need to sit down and discuss your particular project. 
  • Building a Library.  Illumina has sample prep kits for each of their applications.  A lot of labs like to save money by doing their own library prep and we are happy to run these samples but please consult with the sequencing staff as to the requirements.  Low diversity libraries will require a 20-30% PhiX control spike-in to normalize the library and enable better sequencing results. 
    • Each Illumina sample prep kit requires a different amount of starting material and we will advise you as to how much we need depending on your project and library generation needs. 
    •  
  • Sequencing run times vary depending on the application but are usually done within 2-3 days. 
  • Data is typically stored in Illumina's BaseSpace Cloud and subsequently be shared with you via email invite. However, we do have a dedicated server where data can be stored as well.
  • Data analysis - ​The analysis strategy should be discussed during consultation.  You will receive the following files depending on the application.

 

    • Raw data in the form of compressed fastq data files.
    • Aligned reads will provide bam formatted files (.bam) which can be viewed using a Genome Browser such as the Broad's Integrated Genome Viewer (IGV).
    • Variant calling identifies SNPs and short indels and are generated in a Variant Call Format (.vcf) file.
    • Further analysis can be performed by a bioinformatician. We do not have a bioinformatics person on staff but rather have someone we can refer you to who is an independent contractor. His contact information is James White, PhD at james.dna.white@gmail.com or http://jameswhitephd.com. James has extensive experience and references can be supplied.

 

  

 

 

 

 

 

 

 

 

 

Please feel free to contact us: Lab: (410) 955-2739 | Fax: (410) 614-7566 | e-mail: syn_seq@jhmi.edu

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