Best practice for sequencing pooling and QC

Release date : Jan 27,2026

Best practice of Recommended Sequencing Mode:

Although different SeekGene products may specify varying read lengths in their user manuals, we strongly recommend sequencing all libraries using the PE150 mode for optimal data quality and compatibility.

Best practice of Recommended Read Depth and Capture Targets:

Product Type	Recommended Sequencing Depth	Notes
3′ RNA-seq	350–400 million reads per 10,000 cells
5′ RNA-seq	350–400 million reads per 10,000 cells
V(D)J	35 million reads
scFAST-seq	≥400 million reads per 10,000 cells
ATAC + RNA Multi-omics	RNA: ≥400 million reads per 10,000 cells ATAC: ≥400 million reads per 10,000 cells	Sequence RNA and ATAC separately
SeekSpace (Spatial Transcriptomics)	RNA: ≥650 million reads Spatial: ≥150 million reads	Sequence RNA and Spatial separately

Best practice of Recommended Pooing:

SeekGene libraries can be categorized into two groups:

Poly(A)/Poly(T)-rich libraries, such as:

3′ transcriptome libraries, Spatial barcode libraries

Other libraries, such as:

5′ transcriptome, scFAST-seq, ATAC, or V(D)J libraries

Pooling Recommendations for Illumina and GeneMind platforms:

3′ transcriptome or spatial barcode libraries should not exceed 30% of a sequencing lane, otherwise they may impact other libraries’ data quality.
ATAC libraries must not be pooled with Poly(A)/Poly(T)-rich libraries (e.g., 3′ or spatial libraries) because this will severely compromise ATAC data quality.
When pooling ATAC libraries with other compatible types, ensure that ATAC does not exceed 50% of the total lane.
It is acceptable for 3′ and spatial barcode libraries to be sequenced together or to occupy a full lane, but we recommend adding 10% PhiX.
Always pool based on library molarity (nM), not by concentration (ng/µL).

Pooling Recommendations for BGI platform:

Full-length libraries and ATAC libraries are not recommended for sequencing on the BGI platform, especially ATAC libraries.
For all other SeekGene products, no single library type should exceed 10% of a sequencing lane.
Even with PhiX addition, 3′ libraries often show reduced output on BGI.
TCR/BCR libraries also tend to produce lower yields on the BGI platform.

Best practice of Imbalanced Single Cell Library:

Single-cell libraries are base composition–imbalanced and typically exhibit broader fragment peaks.
As long as the library meets QC specifications described in the product manual, it is suitable for sequencing on Illumina or GeneMind platforms.
A broad peak profile does NOT affect sequencing performance on these platforms.

Best practice of Recommended Sequencing Platforms:

Illumina and GeneMind platforms are fully compatible with SeekGene libraries.
BGI platform requires additional phosphorylation and circularization steps prior to sequencing. Reagent kits or support are typically offered by the sequencing service provider.
Other sequencing platforms have not yet been validated by SeekGene. If necessary, please share your library QC report and structure with your sequencing provider for compatibility confirmation.

Best Practice of Pre-QC Read Processing

Before running the QC tools, preprocess sequencing files using fastp to ensure read length consistency.
Add the parameter --length_required 60 to retain reads longer than 60 bp.

Example command:

fastp \

-i FW2024-636-03-T4-TCR_R1_001.clean.fastq.gz \

-I FW2024-636-03-T4-TCR_R2_001.clean.fastq.gz \

-o FW2024-636-03-T4-TCR_R1_001.cleannew.fastq.gz \

-O FW2024-636-03-T4-TCR_R2_001.cleannew.fastq.gz \

-j FW2024-636-03-T4_TCR.json \

-h FW2024-636-03-T4_TCR.html \

--cut_tail_window_size 1 \

--cut_tail_mean_quality 3 \

--cut_tail \

--length_required 60