Library Construction Services
The Translational Genomics Core at Partners HealthCare Personalized Medicine offers a number of library construction services. We welcome the opportunity to discuss projects with potential clients to ensure the selection of an appropriate method for their scope of work. We also pay close attention to changes in researcher focus and technology and welcome discussions with clients and companies for new technology testing and development, collaborative protocol development, and beta-testing of instrumentation and software.
Library construction services include:
- Whole genome
- Whole exome (human)
- Total RNA/mRNA Whole Transcriptome (RNAseq)
- Microbiome 16S rRNA
- Small RNA
We offer personalized target enrichment services, including:
- PCR amplicon based target enrichment
- Commercial Target Enrichment kits
- SureSelect (Agilent)
In addition, we are actively developing and expanding our library construction services, including library construction for methylation profiling by the RRBS method. All projects are managed through GIGPAD, our Laboratory Information Management System (LIMS), and are taken on a first-come, first-served basis, according to sample type and library construction method. Quality control (QC) is carried out for all processes and includes grouping of a PhiX sample with customer samples prior to library construction. All RNA-Seq library construction methods include incorporation of Ambion ERCC spike-in control mixes prior to cDNA synthesis as a QC for the dynamic range of the assay and its limit of detection.
Sample requirements for library construction
Our standard library construction method for most samples uses the Beckman Coulter SPRIworks with NEXTflex adaptors, followed by PCR enrichment and size selection using SAGE Pippin Prep, if needed.
The purity of the sample should be measured by the 260/280 optical density (OD). A gel picture is required to measure the quality of the submitted sample. Please submit samples in either VWR free-standing RNase/DNase free 0.5ml tubes (89004-286) or Fisher RNase/DNase free plates (AB-0800). We can provide these tubes/plates free of charge. If samples are submitted in a different container type, there will be a $5 transfer charge per sample. Please contact us if your DNA or RNA is limiting.
|Service||Sample type||Input range||Solution||Concentration||Quality||LC method|
|Whole Genome/Whole Exome||Genomic DNA||1 ug-5 ug||TE or nuclease free water||50 ng/ul||1.7-2.0 260/280 OD, >1.5 260/230 OD||Standard|
|Whole Transcriptome||Total RNA or mRNA||50 ng-500 ng||DNase and RNase free water*||50 ng/ul||
RIN>7, 1.75-2 260/280 OD, 1.75-2 260/230 OD
|PCR Amplicon||DNA amplicons (>350bp)||50 ng||TE or nuclease free water||15 ng/ul||
1.7-2.0 260/280 OD, >1.5 260/230 OD
*A minimum volume of 10 ul is required for all samples so that enough sample is available for up front QC as well as for library construction.
**RNA samples: If RNA is extracted using TRIZOL or other organic reagents, the final clean-up step must be performed using an RNeasy column cleanup or other similar method. RNA samples should also be prepared using an in solution DNAse treatment. We have found on-column DNase digestion has not been suitable to eliminate all DNA.
Quality control is carried out for all processes and includes grouping of a PhiX sample with customer samples prior to library construction. All libraries are quality controlled by picogreen, using Qubit, and then normalized to 10 nM, followed by qPCR, using P5 and P7 adaptors, to measure the amount of library containing the correct adaptors. The concentration is then further adjusted to produce a sufficient number of clusters in cluster generation. Any library failing QC will be returned to the customer or can be run with no guarantee of read number.
What is a “RIN” score?
A RIN score is the RNA Integrity Number. This number is derived from the Agilent Bioanalyzer or TapeStation testing of RNA. The RIN value ranges between 1 and 10, with 10 being pure non-degraded RNA and 1 being completely degraded RNA. This value is calculated from the Agilent analysis by the Agilent “Expert” Software using the entire scan output from the individual RNA sample. This number is used as a quality metric to aid us in assessment of the integrity of individual RNA samples, independent of the relative concentrations. We use this number to inform investigators in advance of any concerns about inclusion of a sample or samples in an analysis. Values below 6 are considered failed and not likely to provide useful or reproducible results.
We work on a first-come, first-served basis. On average, it takes 2–3 weeks to complete library construction once samples are received. The expected turn-around time increases for development projects and optimization steps.
Ordering and pricing