Sample Submission Guidelines

Illumina and Sample Quality Control

Sample (Nucleic Acid) QC Only

  • Please provide ≥3µl per sample. If requesting both Qubit and Agilent services, please provide ≥4µl.
  • Samples should be delivered in 1.7ml microcentrifuge tubes.
  • If submitting more than 48 samples, samples should be provided in 96-well PCR plates and well location indicated in sample table.
  • Strip tubes will not be accepted.

RNA/ChIP-seq/Amplicon-seq

  • Samples should be delivered in 1.7ml microcentrifuge tubes with minimum volume of 10µl total RNA in RNAse-free water or appropriate elution buffer. RNA should be DNase treated.
  • If submitting more than 48 samples, samples should be provided in 96-well PCR plates and plate location indicated in sample table.
  • Strip tubes will not be accepted.

RNA-Seq Input Recommendations

PolyA selection:

-Requires 5-10 micrograms or more at minimum concentration of 100ng/ul

-Total RNA must be of high integrity (RIN > 8.0)

RiboErase rRNA depletion (Human/Mouse/Rat only):

-Requires 500 nanograms to 1 microgram or more at minimum concentration of 50ng/ul

-Method can be compatible with degraded RNA, so long as each sample within the experiment is similarly degraded

FastSelect rRNA depletion (Human/Mouse/Rat only):

-Requires 500 nanograms to 1 microgram or more at minimum concentration of 50ng/ul

-Method can be compatible with degraded RNA, so long as each sample within the experiment is similarly degraded

FastSelect with rRNA and Globin depletion (whole blood samples only):

-Requires 500 nanograms to 1 microgram or more at minimum concentration of 56ng/ul

-Method can be compatible with degraded RNA, so long as each sample within the experiment is similarly degraded

Clontech SMARTer (a polyA-based amplification approach for low input samples):

-Requires 10 nanograms or more at minimum concentration of 1.1ng/ul

-Total RNA must be of high integrity (RIN > 8.0)

Sigma SeqPlex:

– Requires 10 nanograms or more at minimum concentration of 1.2ng/ul, for FFPE recommended input is 50 nanograms at minimum concentration 6ng/ul

-Method is compatible with degraded RNA, particularly FFPE-derived samples, so long as each sample within the experiment is similarly degraded

-If total RNAs are of good integrity, we recommend Clontech for low input RNA-seq

ChIP-seq

  • Chromatin immunoprecipitated DNA should be submitted in DNAse-free water.
  • Samples should be fragmented to 150-500bp prior to IP.
  • Recommended input for ChIP-seq is 10ng in minimum concentration of 0.33ng/µl.

Amplicon-seq

  • Prior to requesting this service, please discuss your project with the GTAC@MGI to confirm that Illumina sequencing is appropriate for your sample type.
  • PCR amplicons are submitted for Illumina sequencing library preparation. Each sample (typically a pool of amplicons or concatemerized amplicons) will be uniquely dual-indexed.
  • Please Note: The minimum amount of sequencing that can be requested in a shared lane (per submission) on an Illumina NovaSeq6000 S4 flow cell is a target of 50 million read-pairs.

DNA

  • Samples should be delivered in matrix 2D tubes (preferred) or 1.7ml microcentrifuge tubes.
  • Screw top tubes are not compatible with our automated platforms.
  • Strip tubes or plates will not be accepted.
  • Submit in ≥20µl appropriate elution buffer (example: 10mM Tris-HCl).
  • If samples are highly concentrated, please dilute to 25-100 ng/µl.

DNA Input Recommendations

Kapa Hyper PCR-free (automated):

-Requires 600 nanograms to 1 micrograms or more

Kapa Hyper amplified (automated):

-Requires 100 to 500 nanograms or more, coverage dependent

IDT WGS with PCR (manual):

-Requires 5 to 100 nanograms or more

IDT WGBS with PCR (manual):

-Requires 100-200 nanograms or more

IDT WGBS with PCR (automatedl):

-Requires 200 nanograms or more

Prepared Libraries

  • Libraries required to be submitted at 5-30nM in ≥30µl nuclease-free water or buffer (example: Tris-HCl). Submitting libraries outside of these specs may result in additional qPCR charges.
  • If requesting full NovaSeq flow cell, please submit:
    • ≥120µl for SP/S1 flow cell
    • ≥175µl for S2 flow cell
    • ≥330µl for S4 flow cell
  • Samples required to be delivered in matrix 2D tubes (preferred) or 1.7ml microcentrifuge tubes. Tubes must be clearly labeled matching the sample manifest or independently identifiable.
  • Strip tubes will not be accepted.
  • If requesting uneven pooling, please indicate desired ratios in sample sheet.
  • Pre-pooling of libraries is preferred, but not required.

Sample Submission Guidelines: Final Library QC Services (Toni’s lab, correct?)

  • Library pools should be submitted at 10-30nM in ≥30µl nuclease-free water or buffer (example: Tris-HCl).
  • Individual libraries may be submitted at 10-30nM in 10-20µl nuclease-free water or buffer.
  • If requesting full NovaSeq flow cell, please submit:
    • ≥120µl for SP/S1 flow cell
    • ≥175µl for S2 flow cell
    • ≥330µl for S4 flow cell
  • Samples should be delivered in 1.7ml microcentrifuge tubes or 96-well PCR plates.
  • Strip tubes will not be accepted.
  • If requesting uneven pooling, please indicate desired ratios in sample sheet.

10x Genomics

  • Additional QC/Recount will NOT be performed at the GTAC@MGI, the cell/nuclei concentration must be determined by the collaborator providing the samples.
  • Cells/Nuclei must be delivered on ice.

10x Input Recommendations

Nuclei:
  • Standard Assay: 1200 nuc/uL (minimum 700nuc/uL) in 1x PBS with 1% BSA and 0.2U/uL RNase inhibitor in a minimum of 20uL.
  • Please contact Jen Ponce – jgodfrey@wustl.edu for Low and High Throughput options.
Cells:
  • Viability and removal of cell debris is important. The instrument does not distinguish live cells from dead and will package debris as well reducing the overall cell count for your sample.

Visium Spatial

  • FFPE Blocks only
  • BioAnalyzer DV200 >50% requirement
  • Human or Mouse only

Fixed Assay

  • SinglePlex or MultiPlex (1-16 samples)
  • Human or Mouse only
  • 200,000 cells in the SinglePlex configuration
  • 50,000 cells per sample for the MultiPlex configuration

FACSseq/SMARTseq

RNAseq for single cells sorted into individual wells in plates:

Please contact the GTAC@MGI prior to sample preparation regarding specific plate types, lysis buffer, RNase inhibitor, and volumes required for these services.

FACSseq (single-cell lysates in 96-well plate)

This method will provide sequence data that is strongly 3’ biased due to the location and detection of the barcodes added during cDNA generation. Because of this we are only able to provide gene level data, not transcript level.
Please Note: FACSseq libraries are not compatible with our standard dual-indexed NovaSeq S4 (2×150) sequencing workflow. Single indexing and single-end reads are utilized. Sequencing instrumentation and run type will be determined based on the size of the project and desired read-depth. The GTAC@MGI will confirm run type when samples are received. Targeting 1 to 2 million reads per cell is recommended.

SMARTseq2 (single-cell lysates in 96-well plate)

Assuming adequate sample quality (RNA is highly intact) the SMARTseq method generates sequence data across full transcripts, and typically requires greater sequencing depth compared to FACSseq to detect similar numbers of expressed genes per cell. These libraries are dual-indexed and sequenced in our standard Illumina NovaSeq S4 (2×150) workflow. Targeting 5 to 10 million read-pairs per cell is recommended.

new_ilnxgnsq_rna_indsv_facsseq_plates_unprocwells (below field:)

FACSseq and SMARTseq2 library preps are charged by the (96-well) plate. If partial plate is processed, there is no discount for unprocessed wells.

PacBio Sequel II

  • Samples should be delivered in 1.5ml-2mL microcentrifuge tubes (preferred) or matrix2D tubes.
  • Strip tubes and plates will not be accepted.
  • Submit in elution buffer or nuclease-free water.

PacBio Whole Genome Sequencing (CCS/HiFi WGS)

  • 1-2 SMRT Cells: 8ug+ of genomic DNA
  • 3-5 SMRT Cells 16ug+ of genomic DNA
  • If sample isn’t limited submitting additional sample is helpful
  • If sample is limited less stringent size selection is possible. Please discuss with PacBio Production Manager Chris Markovic (cmarkovi@wustl.edu)
  • DNA Purity: 260/280= 1.8-2.0 260/230= 0
  • Genomic DNA Size: Majority of gDNA should be 40kb or greater

PacBio Iso-Seq (RNA-Sequencing)

  • Requires 300ng total RNA in 7uL or less or 160ng-500ng of cDNA in 46uL or less
  • Minimum concentration for total RNA is 43ng/uL. Minimum concentration for cDNA is 3.5ng/uL
  • Minimum RIN value for total RNA is 7.0
  • Please submit few extra uLs if available

PacBio Other Applications (Amplicons, Metagenomic, Multiplexed Microbial, etc)

Sample submission requirements: Contact PacBio Production Manager Chris Markovic (cmarkovi@wustl.edu)

Oxford Nanopore Promethion24

  • Samples should be delivered in 1.5ml-2mL microcentrifuge tubes (preferred) or matrix2D tubes.
  • Strip tubes and plates will not be accepted.
  • Submit in elution buffer or nuclease-free water.

Sample submission requirements: Contact Oxford Nanopore Production Manager Chris Markovic (cmarkovi@wustl.edu)

Bionano Saphyr Optical Genome Mapping

Sample submission requirements: Contact Bionano Production Manager Chris Markovic (cmarkovi@wustl.edu)

 16s MVRSION

  • Samples must be submitted in 96-well PCR plates.
  • Concentration of sample required is dependent on the amount of host contamination present n the samples.
  • Please provide ≥5µl gDNA in DNAse-free water or appropriate DNA elution buffer (example: 10mM Tris-HCl).

Sample submission requirements: Contact Lab Research Manager Chris Sawyer (csawyer@wustl.edu)

Somalogic SomaScan

GTAC@MGI requires all samples be submitted with the correct submission form and template matrix. Samples must be randomized across the 96 sample matrix. Samples received without submission forms, or forms missing required information will not be processed until complete information is provided.

Samples should be provided in well labeled 1.7ml tubes, with the unique label matching the sample name on the submission form.  As well, please include PI and date. Please provide 150 µl aliquots for each sample.

GTAC can accommodate projects in the following manner:
  1. Ideally, samples should be submitted in sets of 85 as these Plasma or Serum SomaScan kits are provided only as 85 sample reaction kits.
  2. Projects of smaller sizes can be submitted for kit sharing; however, the wait time to begin processing is unknown in order to match other small projects to the 85 sample kit runs.
  3. If you cannot fill an entire kit, but do not wish to wait to be matched with another project, we can begin per your approval.  (GTAC can run some samples as technical replicates if sample volume allows.)  Please note that you will be charged for all 85 reactions if you wish to proceed with an entire kit.

NOTE: Improperly prepared samples may impact analyte measurements. In addition, light scattering can be indicative of particulates of sufficient size to clog filters during a SOMAscan Assay run, which could lead to a failed sample run.

Quality control for microarray:

Prior to processing samples through the SomaScan assay, GTAC microarray will evaluate all submitted protein. Please review SomaScan Sample Handling. Initial QC specifications will require an email confirmation if you wish to proceed with processing. If a sample should fail due to a technical error, investigators will only be charged for the samples that were completed.

SomaScan exports and analysis:

Agilent SOMAScan slides will be processed through the Agilent scanner and image data will be extracted using Agilent Feature Extraction software. Extracted initial raw signal intensity data will be uploaded onto the SOMAScan server for data QC and standardization. The final data set will include SOMAscan generated .adat file, a reformatted text file with standardized relative fluorescent unit (RFU) for ~7K proteins, SOMAscan Quality Statements (SQS) and basic differential expression.