Our most popular protocols in brief
- Whole exome sequencing
Purified genomic DNA will be used to generate a library of the exons of nearly all human genes, including many variants of interest. The library will be generated using the NimbleGen SeqCap EZ Human Exome Kit v3.0 from Roche. This kit captures 64 Mb of target, including more than 20,000 genes in the human genome derived from RefSeq RefGene CDS, CCDS, and miRBase v14 databases, plus coverage of 97% Vega, 97% Gencode, and 99% Ensembl databases. Our standard output provides an average of 30x coverage of target regions.
- Targeted exome sequencing of selected regions of interest
Purified genomic DNA will be used to generate an enriched library of the exons and the flanking intronic regions (10 to 30 bp) of the specific genes of interest. The library will be generated using custom probes from Illumina designed using DesignStudio. This method can be used to sequence any genomic region of interest.
- Whole transcriptome sequencing (RNA-Seq)
Total RNA will be used to generate a library of either ribosomal RNA-depleted, or polyA-selected fragments of the entire transcriptome. The library will be generated using Illumina’s TruSeq Stranded Total RNA Library Prep Kit.
- microRNA sequencing (miRNA-Seq)
Total RNA will be used generate a library specifically targeting microRNAs. The library will be generated using Illumina’s TruSeq Small Library Preparation Kit. If you are interested sequencing other small RNAs, please inquire to the MGC.
- Amplicon sequencing
This can include almost any type of DNA fragment, whether it is generated by PCR, or restriction enzyme digestion, or other method. One common application (mentioned above) is targeted exome sequencing, but any population of PCR targets can be sequenced, from a single amplicon to a massively multiplexed population. Plasmid inserts can be sequenced, either as excised fragments or in the context of the whole plasmid. Often NGS sequencing can result in a significant saving or time and/or money over Sanger sequencing.
- Chromatin immunoprecipitation sequencing (ChIP-Seq)
Purified genomic fragments are used for constructing a library. Immunoprecipitated fragments are provided by the investigator, and the library is generated using the Illumina TruSeq ChIP Library Prep Kit.
- Microbiome sequencing
Microbial diversity is measured by sequencing many of the variable regions of the 16S ribosomal RNA sequences present in the population. The investigator provides genomic DNA; it does not have to be pure microbial DNA. The libraries are constructed using the Ion 16S Metagenomics Kit and sequenced on the Ion Torrent PGM.
- Other sequencing protocols
MGC can sequence almost any project that you have in mind. Other sequencing types that we have completed successfully include bisulfate-reduced methylome sequencing, MNase-Seq, and ribosomal profiling; in these cases, libraries were supplied to MGC by the investigator. Many other varieties of sequencing are available, including whole genome sequencing, targeted RNA sequencing (for gene expression analysis), single-cell transcriptome sequencing, microbial whole genome metagenomics, and many others.
- Sequencing machines
Most libraries will be sequenced on either our Illumina HiSeq2500 or MiSeq platforms. Both machines can be operated at a range of scales, depending on the application and number of samples. For select project types, i.e., microbiome sequencing, it may be more appropriate to use our Ion Torrent PGM sequencer.
- Data Analysis
The resulting sequence datasets are subjected to primary and secondary analysis. Typically this involves alignment and initial functional calls. For example, for RNA-Seq, an investigator would get back a spreadsheet of differentially expressed genes along with associated probabilities. For whole exome projects, a variant call file (.vcf) would be returned to the investigator. We will also provide raw output files, and BAM files, if requested. Additional tertiary or other analyses may be performed upon request. We are also open to collaboration for mining of existing sequence data generated by other sources.
Primary analysis aims to assure proper data production and includes alignment of the sequencing data against reference genomes, or custom reference sequences. Initial quality control aims to evaluate data quality of the run as data is being produced on the instrument. Overall evaluation of the quality of the final dataset is additionally performed both pre- and post-alignment and filtering.
Secondary analysis includes realignment and assembly of all the generated sequencing reads, recalibration, variant calling, and additional analysis such as the pathogenic potential of the identified variants. A modular pipeline built around the Broad Institute's GATK best practices is generally employed and adjustable on request.
A limited number of identified variants of interest may be confirmed with bi-directional Sanger sequencing upon request.
After completion of the analysis on the PI-requested genes and confirmation of the variations of interest, the sequence results on the inquired genes will be reported to the requesting PI. Note that the Molecular Genomics Core is not currently CLIA certified, so no results can be used for clinical diagnostic purposes. The PI is responsible for any appropriate patient consent documentation.