Library Services

For the "power user" labs that wish to perform their own library preparation, the GSL will be happy to sequence prepared libraries on any of our platforms (Illumina, 454, SOLiD). The following policies apply for user prepared libraries:

Electropherograms for two libraries prepared by GSL clients. The top library has the expected profile, with a single peak of fragment sizes. The bottom library is likely overamplified, with a secondary peak around twice the size of the expected fragments.

1. User prepared libraries will first be subjected to the same three QC assays that are performed on GSL-prepared libraries:
   • Initial quantification via Qubit
   • Bioanalysis using the Agilent 2100 Bioanalyzer - allows us to check the average library fragment size and test for the presence of multiple peaks. Multiple peaks are a strong indication of poor sequencing performance
   • Final quantification with a Kapa Library Quant Kit - using a 10nM dilution (based on Bioanalysis data), which independently validates concentration and is used to determine final concentration for sequencing
2. Once the QC process has been completed, all QC-related data will be posted to the project page and the GSL will make a recommendation for the most appropriate sequencing conditions
3. The submitting investigator will then make the final decision on the sequencing conditions.

Using the above protocol, the GSL makes every effort to accurately determine library concentration and the appropriate sequencing dilution. However, for libraries created by other labs, there is sometimes difficulty in predicting the appropriate dilution, especially if the libraries have multiple peaks or consist of pooled samples. The GSL will document the quantitation process for the libraries in question and the final decision on sequencing concentration will rest with the user. The GSL cannot guarantee performance or sequencing output for libraries generated by outside users; therefore payment is required prior to sequencing.

Special Library Considerations

• Libraries cannot be indexed (barcoded) after preparation, it must be done as part of the library prep protocol. Therefore please contact us if you are interested in indexing your samples
• The GSL supports several forms of indexing, including Illumina Tru-Seq and Nextera
• Not all forms of indexing or library preparation are appropriate for current sequencer software versions. The user is responsible for disclosing all relevant library prep details before sequencing is attempted to avoid disappointment in data output. For example, certain library construction protocols can result in identical sequences for the first several bases of each read, which causes problems for the software when it tries to define the cluster locations. Previous versions of the Illumina software could defer basecalling for a given number of cycles to avoid this problem, but this capability no longer exists. For such libraries, the only options for accurately sequencing such libraries are
  1. mix with another library from the same user and demultiplex reads after sequencing
  2. mix with Phix and lose a fraction of the usable reads
  3. sequence at a low cluster density so that the clusters are spatially separate from each other

Sample Submission Requirements

Libraries should be submitted at 10nM concentration in 10mM Tris (pH 8.0), and 10µl should be provided for ease of handling. If you provide bioanalysis profiles and flourescence quantitation values, we can skip those steps during QC. Libraries which are not at 10nM will be subject to an additional handling fee. When libraries are submitted, please provide as much information about their construction as possible, as it helps us provide you with the data you expect.

GSL Data

Data output for library submission includes, at a minimum, fastq files. These will be demultiplexed for an additional fee if a standard indexing kit is used which requires a third indexed read (such as Illumina Tru-Seq or Nextera). Additional analysis, such as alignment to a standard genome, can also be requested. Due to the size of sequencing data, if an alignment is done, only .bam files (and the .bai or bam index file) are provided as results. Fastq files are not also provided due to the redundancy of data. Users wishing to perform their own analysis of the raw data can easily re-generate the original fastq files using the bam2fastq program.