Library construction is the process of taking DNA or RNA from its "native" state to a form that can be run on the Illumina sequencers. For RNA samples this usually involves a purification of mature mRNA or a depletion of ribosomal RNA, followed by cDNA synthesis, adapter ligation and indexing, and a bead cleanup to remove excess primers and other undesirable material. For DNA samples, this might involve a fragmentation if the material is >800bp, adapter ligation and indexing, and a bead cleanup. Some protocols also involve a size-selection step.
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 which improve turnaround time, cost, and library uniformity. The BPF has used the Sciclone NGS G3 Workstation liquid handler for years, and recently added the SPT LabTech Firefly to our instrumentation.
We offer a variety of library methods; Please see the Library Prep Options section below for a list of methods currently available from our core. Pricing per sample varies, and becomes less expensive with bigger batches of samples. For free pricing estimates, please email us with some details about your project at nextgen@genome.med.harvard.edu. 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 (self-run)
- MJ Research Tetrad DNA Engine Thermal Cycler
- Applied Biosystems Veriti Thermal Cyclers
- Revvity FlexDrop iQ
- Revvity (Perkin Elmer) Sciclone NGS G3 Workstation
- SPT Labtech FireFly
Please provide starting material as specified in the table below. We recommend ordering TapeStation services prior to ordering your library prep service.
DNA options
| Sample Type | Prep Type | Minimum Input Material Amount |
|---|---|---|
| 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 |
| NEBNext Ultra Express DNA A streamlined, faster workflow allowing for quicker turnaround times. End-repair, A-tailing, and Adapter Ligation are followed by PCR and a final PCR-Clean Up | 10 ng | |
| 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 |
| NEBNext Ultra Express FS DNA Similar to the UltraExpress DNA but featuring an enzymatic fragmentation step at the beginning of the protocol. Streamlined protocol allows for rapid turnaround time. | 10 ng | |
| NEBNext Ultra II FS PCR Free DNA High Input An amplification free workflow for DNA-seq based on the standard NEB Ultra II FS workflow. High-yield, high-quality libraries without PCR bias. If you have enough material, we recommend using the high input workflow. | 100 ng | |
| NEBNext Ultra II FS PCR Free DNA Low Input An amplification free workflow for DNA-seq based on the standard NEB Ultra II FS workflow. High-yield, high-quality libraries without PCR bias.This is a lower input workflow for those unable to obtain sufficient sample for the high input protocol. This may result in shorter fragments, lower mapping quality, and higher adapter dimer levels. | 50 ng | |
| 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 |
RNA options
| Sample Type | Prep Type | Minimum Input Material Amount |
|---|---|---|
| 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 |
| 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 |
| NEBNextUltraExpress RNA with polyA mRNA Magnetic Isolation Module Streamlined RNA library prep workflow, allowing for the generation of high-quality RNA directional libraries in a single day. This version of the method is used with the poly(A) mRNA magnetic isolation module which employs an oligo dT-based mRNA isolation ideal for enrichment of mRNA and separation from rRNA when samples are eukaryotic and have intact poly(A) tails | 25 ng | |
| 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 |
| NEBNext UltraExpress RNA with rRNA Depletion Streamlined RNA library prep workflow, allowing for the generation of high-quality RNA directional libraries in a single day. This version of the express workflow begins with the rRNA depletion module to remove ribosomal RNAs employing an RNaseH-based method. The rRNA depleted RNA is then | 25 ng | |
| Coming Soon: Takara SMART-Seq Total RNA Pico Input with UMIs (ZapR Mammalian), for low input with ribo-depletion projects | 250 pg | |
| Total-RNA; (smRNA-seq & miRNA-seq) | Coming Soon: 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 |
NGS-Based Proteomics options
| Sample Type | Prep Type | Minimum Input Material Amount |
|---|---|---|
| Plasma, Serum | OLink Reveal Assay This assay uses DNA-tagged antibodies to identify >1,000 proteins in plasma or serum. | 40 uL Plasma or Serum |
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. . We offer free assistance writing the relevant parts of your Methods section, for any project on which we have collaborated.
Every library we produce is run on an Agilent 4200 TapeStation High Sensitivity D5000 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.
Whether we have prepped the libraries, or you wish to submit libraries ready-to-sequence, we can help with the sequencing portion of the workflow!
Please visit our Next-Gen Sequencing page for more information