Pooling and Submission of Sequencing Libraries
- Only pool samples for sequencing that have compatible i5/p5 and i7/p7 barcodes. More information on Troutman lab barcode collisions can be found here.
- Don’t unknowingly pool samples that have very different molecular weights.
- Typically we target 15-25 million reads per sample for ATAC-seq, ChIP-seq and RNA-seq.
- Samples profiling allelic biases (F1 hybrids) may require 50-100 million reads per sample.
- Follow the instructions in the pooling calculator generated by the BadDNA research group at the University of Georgia.
- Example pooling sheet (coming soon).
- Pooling requires an estimate of each sample’s molecular weight and a qubit quantification of each samples concentration in ng/µl.
Verify the appropriate orientation of the index sequence matches the requirements of the targeted sequencing platform. This must be appropriately entered in the submission sample sheet or demultiplexing will fail. Illumina NovaSeq v1.0 and v1.5 workflows utilize different strategies and require entering i5 index sequences appropriately. Forward strand i5 workflow is performed with v1.0 reagent kits, while reverse complement i5 workflow is performed with v1.5 reagent kits (NovaSeq). For the Illumina MiniSeq or NextSeq platforms, enter the i5 index as the reverse complement. When in doubt, verify with the core facility or sequencing provider and/or read the user manual!!
CCHMC Sequencing
The local CCHMC Genomics Sequencing Facility is currently directed by Hassan Chaib, PhD, and managed by David Fletcher and direct.
- Non-core generated libraries can only be sequenced on a complete purchased flow cells.
- One lane of a NovaSeq X Plus 1-B flow cell configured at PE50 is $1,204.75, per DNA Core Price Calculator.
- More information on submission can be found at the bottom of this page.
Core sequencing options:
Instrument Type Flow Cell Type Number Lanes Flow Cell Total Cycles Expected Output (M Clusters Lane) Expected Output (M Clusters Total)
--------------- -------------------------- --------------- ----------------------- -------------- ---------------------------------- -----------------------------------
iSeq 100 Reverse Complement i1 1 300 4 4
MiSeq Forward MiSeq 1 300, 500, 600 15 15
NovaSeq 6000 Reverse Complement (v1.5) SP (S prime) 1 100, 300, 500 720 720
NovaSeq 6000 Reverse Complement (v1.5) S2 2 100, 200 720 1440
NovaSeq 6000 Reverse Complement (v1.5) S4 2 100, 200, 300 1600 3200
NovaSeq 6000 Reverse Complement (v1.5) S4 4 200, 300 2000 8000
NovaSeq X Plus Reverse Complement 1.5B 2 100, 200, 300 720 1440
NovaSeq X Plus Reverse Complement 10B 8 100, 200, 300 1200 9600
NovaSeq X Plus Reverse Complement 25B 8 300 3000 24000Lane submission requirements:
Service Input.Material Recommended.volume..µl. Diluent Minimum.Concentration
------------------- --------------------------------- ------------------------ --------------- ----------------------
iSeq 100 Illumina-compatible library pool 30 10 mM Tris-HCl 5 nM
MiSeq Illumina-compatible library pool 30 10 mM Tris-HCl 5 nM
NovaSeq SP, S1, S2 Illumina-compatible library pool 40 10 mM Tris-HCl 5 nM
NovaSeq S4 Illumina-compatible library pool 80 10 mM Tris-HCl 5 nM
NovaSeqX 1.5B, 10B Illumina-compatible library pool 30 10 mM Tris-HCl 5 nM
NovaSeqX 25B Illumina-compatible library pool 60 10 mM Tris-HCl 5 nM
CCMHC Submission Sample Sheet:
Use the lab standardized naming convention!