Sample Requirements

Online Submission (Yale only)            Non-Yale, please email.

If you would like YCGA to prepare samples for sequencing, please see appropriate sample submission requirements below.

If you would like to pool/multiplex/barcode samples to be sequenced in the same flowcell lane, please contact our facility for recommendations on how many samples should be run together. The number of samples to pool will depend on your application and flowcell lane cluster densities. While we strive for a fairly even distribution for each barcode, we cannot guarantee this. When given a high quality sample, we can guarantee a minimum of 170 million clusters per lane. Thank you for understanding.

User-prepared Libraries

If you prepare your own libraries and would like only cluster generation and sequencing services, at least 15uL of undiluted library (>20nM) suspended in EB Buffer should be submitted. Please ensure that if you are a Yale user submitting a request form through your WikiLIMS account, that you only select “Library already prepared” from the drop-down menu for the “Sample type.” YCGA provides several sample preparation services, selecting any other “Sample type” option may result in your libraries being delivered to an improper area of the lab and will result in a delay of turnaround time.

All requests for user-prepared samples should include index information submitted in an Excel sheet, with 3 columns in the following order: Sample name, i7 index sequence and i5 index sequence.  A row with this information should be filled out for each submitted library (as applicable).  Please do not provide YCGA with the primer sequence. This Excel sheet can be uploaded under the “Supporting documents” section while filling out the request form or emailed to Christopher.Castaldi@yale.edu along with the RQ number to be uploaded to an existing request form.

Kindly inform us if the DNA was cut with any restriction enzyme, had overhangs before end repair, or has low base diversity.

Libraries with low base diversity will result in lane sequencing clusters to fail and data to be discarded.  There are ways to enhance lane diversity without altering a library, so it is extremely important to convey that your libraries may have low base diversity so YCGA can make accommodations.

If your libraries require a custom primer, please provide YCGA with 20uL of your custom primer at 100uM.

We require inhouse quantitation by real-time PCR as an additional service. YCGA has standardized the required concentrations of libraries for optimal cluster generation using YCGA-prepared sequence libraries. We cannot guarantee optimal cluster densities with user-prepared libraries at our standardized picomolar concentration of DNA. Although many user-prepared libraries come close to optimal cluster numbers, some deviate significantly by giving either very low or very high clusters, compromising the number of mappable filtered clusters. In these situations, we still bill for these sub-optimal runs.

If you prepare your own libraries and would like only QC information, at least 3uL of sample should be provided.

Please ensure that if you are a Yale user submitting a request form through your WikiLIMS account, that you only select “Sample QC Only” from the drop-down menu for the “Sample type.”

Genomic DNA

1 – 5 ug of Purified DNA (5ug recommended) suspended in TE Buffer in a volume not to exceed 50 ul. DNA should be as intact as possible, with an OD260/280 ratio of 1.8-2.

Targeted Exome Capture (Seq-cap)

1.5 – 5 ug of Purified DNA (5ug recommended) suspended in TE Buffer in a volume not to exceed 50 ul. DNA should be as intact as possible, with an OD260/280 ratio of 1.8-2.

Whole Transcriptome (mRNA-Seq) Profiling

500ng-1ug of Purified total RNA suspended in nuclease-free H2O in a volume less than, or equal to, 50ul. RNA should be as intact as possible, with OD260/280 ratio of 1.8-2 and an OD260/230 ratio of 1.8-2. RNA integrity should be verified via an Agilent Technologies 2100 Bioanalyzer or 1% agarose gel. High-quality RNA will show a 28s rRNA band twice the intensity of the 18S rRNA band. For poly A based library preps the RNA should have a bioanalyzer RIN score of 7 or higher.  For samples with low input amount of RNA please contact YCGA for additional options.

Small RNA Querying

500ng of Purified RNA suspended in nuclease-free H2O in 10 ul volume. RNA should be isolated using a technique that preserves small RNAs (using Qiagen’s miRNeasy kit, for example). RNA should be as intact as possible, with OD260/280 ratio of 1.8-2 and an OD260/230 ratio of 1.8-2. RNA integrity should be verified via an Agilent Technologies 2100 Bioanalyzer or 1% agarose gel. High-quality RNA will show a 28s rRNA band twice the intensity of the 18S rRNA band.

ChIP-Enriched DNA Sequencing

10 ng of purified, ChIP-enriched, qPCR-verified DNA suspended in 30 ul TE Buffer should be submitted.