> For the complete documentation index, see [llms.txt](https://knowledge.illumina.com/llms.txt). Markdown versions of documentation pages are available by appending `.md` to page URLs; this page is available as [Markdown](https://knowledge.illumina.com/library-preparation/general/library-preparation-general-reference_material-list/000010216.md).
# Read Depth Requirements for Illumina Rapid RNA kits
The sequencing read depth and read lengths are updated for Rapid kits compared to the non-Rapid versions. For Rapid kits, see the Product Pages, for additional guidance on sequencing depth.
**Note**: M = million
**Rapid mRNA Project Recommendations:** either targeting 10M or 25M clusters/sample
Read length of 2x50 bp or 1x100 bp for Gene Expression on 100 cycle kits
MiSeq i100 Samples per flow cell:
* 10-25M clusters per sample: 25M: 1-2, 50M: 1-5, 100M: 1-10
NextSeq 1000/2000 Samples per flow cell:
* 10M clusters per sample: P1: 10, P2: 40, P3: 120, P4: 180
* 25M clusters per sample: P1: 4, P2: 16, P3: 48, P4: 72
NovaSeq X: Samples per flow cell
* 10M clusters per sample: 1.5B: 150 (75/lane), 5B: 500 (62/lane), 10B: 1000 (125/lane), 25B: 2500 (312/lane)
* 25M clusters per sample: 1.5B: 60 (30/lane), 5B: 200 (25/lane), 10B: 400 (50/lane), 25B: 1000 (125/lane)
**Rapid Total RNA Project Recommendations:** either targeting 25M or 50M clusters/sample
Read length of 2x50 bp or 1x100 bp for Gene Expression on 100 cycle kits, 2x100 bp for Variant detection on 200 cycle kits.
MiSeq i100 Samples per flow cell:
* 25M clusters/sample: 50M: 2, 100M: 4
* 50M clusters/sample: 50M: 1, 100M: 1-2
NextSeq 2000 Samples per flow cell
* 25M clusters/sample: P1: 4, P2: 16, P3: 48, P4: 72
* 50M clusters/sample: P1: 2, P2: 8, P3: 24, P4: 36
NovaSeqX Samples per flow cell
* 25M clusters/sample: 1.5B: 60 (30/lane), 5B: 200 (25/lane), 10B: 400 (50/lane), 25B: 1000 (125/lane)
* 50M clusters/sample: 1.5B: 30 (15/lane), 5B: 100 (12/lane), 10B: 200 (25/lane), 25B: 500 (62/lane)
**Rapid RNA Prep with Exome 2.5 Enrichment Panel Project Recommendations:** targeting 25M clusters/sample
Read length of 2x50 bp or 1x100 bp for Gene Expression on 100 cycle kits
* 2x100 bp for Variant detection on 200 cycle kits.
MiSeq i100 Samples per flow cell:
* 25M clusters per sample: 25M: 1, 50M: 2, 100M: 4
NextSeq 1000/2000 Samples per flow cell:
* 25M clusters per sample: P1: 4, P2: 16, P3: 48, P4: 72
NovaSeq X: Samples per flow cell
* 25M clusters per sample: 1.5B: 60 (30/lane), 5B: 200 (25/lane), 10B: 400 (50/lane), 25B: 1000 (125/lane)
**Rapid RNA Enrichment with VSPv2, RPIP, or RVEK/RVOPv2 Project Recommendations:**
Read depth recommendations for Rapid VSPv2, RPIP, or RVEK kits are unchanged from their non-Rapid versions: Illumina recommends starting with 1 million clusters per sample, and optimizing as necessary.
**Rapid RNA Prep with Enrichment with Custom Enrichment Panel v2:**
Here is general guidance on target reads/gene for variable panel sizes: For Illumina RNA Enrichment with Custom panels, the target number of reads per sample depends upon the panel size and data analysis goals.
* For detection of low frequency fusion or Small Nucleotide Variants (SNVs) in a somatic tumor sample Illumina recommends targeting about 500x coverage of the target mRNA transcript region, or about 5000 reads per gene, assuming an average mRNA transcript length of 2000 bp. This high depth of coverage allows for detection of low frequency variants and accounts for variation in gene expression across the gene panel targets in the sample.
* For Gene Expression profiling Illumina recommends targeting about 1000-2000 clusters per gene target.
Example: Low Frequency Fusion or SNV (target 500x):
For a 100 gene panel targeting 500x coverage of the mRNA transcripts averaging 2000 bp per gene with a 2x100 bp read length, would require 500,000 clusters per sample. Actual performance may vary depending on gene expression in the target samples.
* 100 genes x 2000 bp per gene x 500X coverage = 100 Mb
* For 2x100 bp reads, or 200 bp per cluster, 100 Mb/200 bp= **500,000 reads (clusters) per sample**
* In terms of reads per gene 500,000 clusters / 100 genes = 5000 reads/gene
Example: Gene Expression Profiling (target 2000 reads per gene):
For a 100 gene panel targeting 2000 reads per gene of the mRNA transcripts with a 2x100 bp read length, would require 200,000 clusters per sample. Actual performance may vary depending on gene expression in the target samples.
* 100 genes x 2000 reads per gene = 2**00,000 reads (clusters) per sample**
Users may choose to target higher depth to detect somatic variants at low Variant Allele Frequency (VAF). See these examples below.
Fusion detection examples from Oncology Panels
Example: TruSight Fusion (507 genes; 3 million reads/sample) = 6000 reads/gene
Example: 109 gene panel in RRPE datasheet sequenced at 800k reads= 7,300 reads/gene
TSO500 RNA Panel for Fusions: 44 genes, 5M reads= 113k reads/gene with 2-5% VAF detection of variants.
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| *For any feedback or questions regarding this article (Illumina Knowledge Article #10216), contact Illumina Technical Support* [*techsupport@illumina.com*](mailto:techsupport@illumina.com?subject=Question%2FFeedback%20Regarding%20Illumina%20Knowledge%20Article%20#000010216%20-%20Library%20Preparation%20\&body=Dear%20Illumina%20Technical%20Support,%0D%0A%0D%0A)*.* |
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