Library construction is the process of taking DNA or RNA from its "native" state to a form that can be run on the Illumina sequencer. The majority of protocols require fragmentation of the nucleic acid material to a specific size range. The BPF offers enzymatic fragmentation, as well as shearing services using the Covaris M220 instrument. There are dozens of methods and kits for the production of Illumina compatible libraries available as well as multiple automation platforms to run these methods. The BPF has adopted the Apollo Wafergen 324 and the Sciclone NGS G3 Workstation. We offer a variety of library methods on this instrument. Please see the Library Prep Options section below for a complete list of methods currently available from our core. If you are interested in a library prep service and do not see it below, please contact us. We are always happy to discuss options not yet listed!
- Covaris M220
- Eppendorf Mastercycler ep Gradient S 96
- MJ Research Tetrad DNA Engine Thermal Cycler
- Qiagen Qiagility Robot
- Revvity FlexDrop iQ
- Revvity (Perkin Elmer) Sciclone NGS G3 Workstation
- SPT Labtech FireFly
Please provide starting material as specified in the table below. If you are not able to determine the quantity or quality of your starting material, you should order Bioanalyzer or TapeStation services prior to ordering your library prep service.
DNA options
| Sample Type | Prep Type | Minimum Input Material Amount | Pricing Information |
|---|---|---|---|
| Amplicon/PCR Products and ChIP < 800bp are ideal; All DNA subtypes are compatible | KAPA HyperPlus Enzymatic shearing is involved as needed, followed by End-Repair, A-Tailing, and Adapter Ligation. A final PCR-Clean removes dimer products. This chemistry has been noted as particularly helpful for challenging samples such as FFPE DNA. | 1 ng | Cost guide for HyperPrep+ here |
| cDNA and gDNA > 800bp | Illumina NexteraXTA transposase with sequencing adapters is used to fragment the DNA into appropriate sizing for sequencing. Unique indices are then added via PCR, and a final PCR Clean-Up process removes adapter dimer. |
1 ng | Cost guide for NexteraXT here |
| Microbial Genomic DNA | Illumina 16S The V3 and v4 regions are amplified and purified. Illumina sequencing adapters with dual-indexing barcodes are added, and a final purification is performed. Sequencing requires 40%-50% PhiX to account for low diversity. We can accommodate other regions if primers are supplied. | 12.5 ng | Cost guide for 16S here |
RNA options
| Sample Type | Prep Type | Minimum Input Material Amount | Pricing Information |
|---|---|---|---|
| Total-RNA; (mRNA-Seq) | Low-Input mRNAmRNA pull-down and cDNA synthesis is performed using the Takara SMARTseq v4 kit, followed by library prep using the Illumina NexteraXT kit. | 1 ng | Cost guide for V4 + NexteraXT here |
| Total-RNA; (mRNA-Seq) | KAPA mRNA HyperPrepStandard mRNA Isolation & Prep (KAPA) Uses the KAPA mRNA HyperPrep kit. mRNA is pulled down using oligo-dT beads, and the resulting mRNA is converted into cDNA. The resulting cDNA then becomes a library through adapter ligation and post-PCR Cleanup. |
50 ng | Cost guide for KAPA mRNA HyperPrep here |
| Total-RNA; (RNA-seq with rRNA-depletion (will prep mRNA and lnc-RNA)) | KAPA HyperPrep with Ribo-Erase Standard Ribo-Depletion & Prep (KAPA)Uses the KAPA HyperPrep RNA & Ribo-Erase kit. rRNA is hybridized to probes and digested with enzymes. The remaining RNA is converted into cDNA. The resulting cDNA then becomes a library through adapter ligation and post-PCR Cleanup. | 30 ng | Cost Guide for KAPA HyperPrep & Ribo-Erase here |
| Total-RNA; (smRNA-seq & miRNA-seq) | Qiagen smRNA/miRNAQiagen's smRNA kit allows for gel-free isolation and prep of RNAs smaller than ˜70bp. The prep incorporates Unique Molecular Identifiers (UMIs) to quantify individual RNA molecules. | 1 ng | Cost guide for Qiagen's smRNA prep here |
Although we provide copies of protocols, we recommend you check the Illumina or other vendor's web sites to obtain the latest version of any given protocol.
Every library we produce is run on an Agilent 2200 TapeStation D1000 HS ScreenTape to assess concentration and size distribution of the library molecules. In addition, a QPCR assay is run to determine functional concentration of the final library in an effort to obtain optimal cluster densities prior to the sequencing process.
Quality Control services have a small additional fee. Further information regarding this service can be found in out NextGen Sequencing section.