Next Generation Sequencing
MiSeq Sequencing:
The Steps to generating a sequence include the following:
- Consultation to discuss your particular project
- Building a library – There are a variety of sample prep kits that can be used
- Running your samples on the MiSeq – run times vary depending on the application but are usually done within 2-3 days.
- Data sharing – data is typically stored on Illumina’s BaseSpace Cloud and subsequently shared with you via email invite. You have to sign up for a BaseSpace account. The run will then be transferred to you.
- 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 be provided as 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 we can refer you to an independent contractor who we have used on numerous occasions. His contact information is James White, Ph.D. and he can be reached at jwhite@respherabio.com
We partner with the Genomic Analysis and Sequencing Core Facility located in the Bloomberg School of Public Health. They have extensive experience with DNA/RNA extraction and library preparation.
Cost of doing a run on the MiSeq:
- The cost of the sequencing kit plus shipping
- A run fee of $500
- Library preparation fees which vary depending on the type and number of libraries generated. You will receive a breakdown of the individual costs associated with each project rather than just receiving a generic library preparation cost.
Cost of sequencing libraries made by you:
Do you want to save money by preparing your own libraries? GREAT! We can help you get the run done usually within a few days.
- The cost of the sequencing kit plus shipping
- A run fee of $500
Some Useful Information:
| Sequencing Kits | Potential Number of Reads |
|---|---|
| MiSeq v3 Kits | ~ 25 million reads |
| MiSeq v2 Kits | ~15 million reads |
| Miseq Nano Kits | ~ 1 million reads |
Calculating Fold Coverage:
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
As an example, sequencing a 5.4 Mb bacterial genome using a 2 x 300 v3 kit that yields approximately 25 million reads
C = (600)(25,000,000)/5,400,000 = 2777 coverage
If you wanted 80X coverage per sample, you could multiplex 34 samples in the run
Please feel free to contact us: Lab: (410) 955-2739 | e-mail: syn_seq@jhmi.edu