RNAseek – Analysis Submission

Submission Name Required A short, descriptive label for this analysis run. Use something you'll recognise later, e.g. "GSE12345_DrugA_vs_DMSO".

Starting Point Required Choose the file type you are starting from. FASTQ runs the full pipeline (QC → alignment → quantification → statistics). BAM/CRAM skips alignment. Count Matrix skips directly to differential expression.

FASTQ

Full pipeline: QC, alignment, quantification & statistics.

FastQC HISAT2 DESeq2

Alignment BAM/CRAM

Skip QC & alignment. Start from aligned reads.

featureCounts DESeq2

Count Matrix

Skip directly to differential expression statistics.

DESeq2

Initialize Your Analysis

Name your submission, select your data starting point, and choose your sequencing assay type to configure the optimal pipeline track.

Multi-assay support Adaptive pipeline Smart routing

Library Type Required Single-end: One FASTQ file per sample. Paired-end: Two FASTQ files (R1 + R2) per sample. Check your sequencing provider's report if unsure.

Single-End

One FASTQ per sample.

Paired-End

R1 + R2 per sample.

Paired-end naming:
Files must share a prefix with _R1/_R2 or _1/_2 suffixes.

Strandedness Unstranded: No strand-specific protocol used. Most common.
fr-firststrand: dUTP/Illumina TruSeq stranded protocol. First read maps to the reverse complement.
fr-secondstrand: Ligation-based. First read maps to the transcript strand. Required by most downstream tools.

FASTQ Files Required Drag and drop gzip-compressed FASTQ files (`.fq.gz` or `.fastq.gz`). For paired-end data, include both R1 and R2 files — they will be matched automatically by filename prefix.

Drag & drop .fq.gz / .fastq.gz or click to browse

Upload Pro Tip

Auto-Pairing: For paired-end data, ensure files share the exact same prefix before the _R1 / _R2 (or _1 / _2) suffixes. The system will group them automatically!

Large Files? No problem. Data is securely uploaded in 50 MB chunks. If your network drops, the upload will automatically resume where it left off.

Sequencing Details & File Upload

Configure your library type and strandedness, then upload your sequencing files. Chunked uploads support files of any size.

50 MB chunked upload Drag & drop Multi-file support Automatic retry

Reference Genome Required Select the reference genome matching your organism. The pipeline uses pre-built indices optimized for your specific assay type's aligner.

Quant Level For RNA-seq, choose Gene-level (default) or transcript-level for isoform-resolution. Other assays (like ChIP-seq) will handle this automatically.

Pipeline Tip

The pipeline automatically selects the best aligner (e.g., HISAT2, Bowtie2, or Bismark) based on the Assay Type you chose in Step 1.

Selecting a standard reference genome uses pre-built indices, which allows your pipeline to begin alignment instantly without indexing delays.

Note: The Quant Level option primarily applies to RNA-seq assays. For other assays like ChIP-seq, quantification defaults to peak-level automatically.

Custom Genome Configuration

Genome Name A unique identifier for your custom genome. Use the organism name and assembly version, e.g. "Sorghum_v3".

Reference FASTA The reference genome sequence file. Accepted formats: .fa, .fasta, and their gzipped or zipped variants.

Upload .fa, .fasta, .fa.gz, or .fa.zip

Browse No file chosen

Annotation GTF/GFF A gene annotation file in GTF or GFF3 format. Required to assign aligned reads to specific genomic features.

Required for standard transcript quantification.

Browse No file chosen

Reference Genome Selection

Choose from pre-indexed genomes for instant alignment, or upload your own custom genome with FASTA and GTF/GFF annotation files.

Human (GRCh38 / hg38) Mouse (GRCm39 / mm39) Mouse (GRCm38 / mm10) Rat (mRatBN7.2 / rn7) Zebrafish (GRCz11) Chicken (GRCg6a) Pig (Sscrofa11.1) Drosophila (BDGP6 / dm6) C. elegans (WBcel235) Yeast (R64-1-1 / sacCer3) Arabidopsis (TAIR10) Custom Genome

Metadata Input Required

Upload CSV

Upload a sample metadata CSV/TSV file.

Manual Edit

Define columns & assign values per sample.

Metadata CSV Upload a CSV or TSV file where the first column is sample (matching filenames without extension) and subsequent columns describe experimental variables such as condition, batch, or treatment.

Drop your metadata .csv or click to browse

Expected CSV Format

sample	condition	batch
Sample1	treated	A
Sample2	treated	B
Sample3	control	A
Sample4	control	B

Format: First column named sample matching filenames without extension.

Column Roles

Primary Group The main biological variable (e.g. condition). Defines groups for DESeq2 comparison.

Batch Effect Optional. Technical variable for ComBat-seq batch correction.

Covariates

Define Comparisons Multi-group DESeq2

Your primary group has more than 2 levels. Define which pairwise comparisons DESeq2 should test.

Experimental Design & Metadata

Upload a CSV or switch to Manual Edit to define your experimental design. Map columns to roles and set pairwise comparisons.

CSV Upload Manual Builder Column Roles Contrast Builder

Significance Thresholds

Adjusted P-value Cutoff Benjamini-Hochberg FDR threshold. Genes with padj below this are considered significant. Default: 0.05. FDR threshold

Min Log2FC Down-regulation cutoff. Genes with log2FC below this value are flagged as significantly down-regulated on the volcano plot. Down-regulation cutoff

Max Log2FC Up-regulation cutoff. Genes with log2FC above this value are flagged as significantly up-regulated on the volcano plot. Up-regulation cutoff

Genes with |log2FC| ≥ 1.0 and padj ≤ 0.05 flagged as significant.

Submission Checklist

Submission Name

Library Type

Data Files

Reference Genome

Metadata

Column Mapping

Statistical Analysis & Visualization

Configure significance thresholds for differential expression analysis. These cutoffs determine how genes are highlighted on volcano plots, MA plots, and heatmaps.

Volcano Plot MA Plot Heatmap PCA DEG Table

RNAseek — Pipeline Submission

Submission Name Required A short, descriptive label for this analysis run. Use something you'll recognise later, e.g. "GSE12345_DrugA_vs_DMSO".

Starting Point Required Choose the file type you are starting from. FASTQ runs the full pipeline (QC → alignment → quantification → statistics). BAM/CRAM skips alignment. Count Matrix skips directly to differential expression.

Assay Type Required Select the sequencing assay used to generate your data. This determines the aligner, reference database, and downstream analysis tools the pipeline will use.

Initialize Your Analysis

Library Type Required Single-end: One FASTQ file per sample. Paired-end: Two FASTQ files (R1 + R2) per sample. Check your sequencing provider's report if unsure.

Strandedness Unstranded: No strand-specific protocol used. Most common.
fr-firststrand: dUTP/Illumina TruSeq stranded protocol. First read maps to the reverse complement.
fr-secondstrand: Ligation-based. First read maps to the transcript strand. Required by most downstream tools.

FASTQ Files Required Drag and drop gzip-compressed FASTQ files (`.fq.gz` or `.fastq.gz`). For paired-end data, include both R1 and R2 files — they will be matched automatically by filename prefix.

Strandedness

Library Type

BAM/CRAM Files Required

Count Matrix Required Upload a CSV/TSV with gene IDs in the first column and raw integer counts. Do not upload TPM/FPKM.

Upload Pro Tip

Sequencing Details & File Upload

Reference Genome Required Select the reference genome matching your organism. The pipeline uses pre-built indices optimized for your specific assay type's aligner.

Quant Level For RNA-seq, choose Gene-level (default) or transcript-level for isoform-resolution. Other assays (like ChIP-seq) will handle this automatically.

Pipeline Tip

Custom Genome Configuration

Reference Genome Selection

Metadata Input Required

Column Roles

Define Comparisons Multi-group DESeq2

Experimental Design & Metadata

Significance Thresholds

Submission Checklist

Statistical Analysis & Visualization

RNAseek — Pipeline Submission

Submission Name *Required A short, descriptive label for this analysis run. Use something you'll recognise later, e.g. "GSE12345_DrugA_vs_DMSO".

Starting Point *Required Choose the file type you are starting from. FASTQ runs the full pipeline (QC → alignment → quantification → statistics). BAM/CRAM skips alignment. Count Matrix skips directly to differential expression.

Assay Type *Required Select the sequencing assay used to generate your data. This determines the aligner, reference database, and downstream analysis tools the pipeline will use.

Initialize Your Analysis

Library Type *Required Single-end: One FASTQ file per sample. Paired-end: Two FASTQ files (R1 + R2) per sample. Check your sequencing provider's report if unsure.

Strandedness Unstranded: No strand-specific protocol used. Most common. fr-firststrand: dUTP/Illumina TruSeq stranded protocol. First read maps to the reverse complement. fr-secondstrand: Ligation-based. First read maps to the transcript strand. Required by most downstream tools.

FASTQ Files *Required Drag and drop gzip-compressed FASTQ files (.fq.gz or .fastq.gz). For paired-end data, include both R1 and R2 files — they will be matched automatically by filename prefix.

Strandedness

Library Type

BAM/CRAM Files *Required

Count Matrix *Required Upload a CSV/TSV with gene IDs in the first column and raw integer counts. Do not upload TPM/FPKM.

Upload Pro Tip

Sequencing Details & File Upload

Reference Genome *Required Select the reference genome matching your organism. The pipeline uses pre-built indices optimized for your specific assay type's aligner.

Quant Level For RNA-seq, choose Gene-level (default) or transcript-level for isoform-resolution. Other assays (like ChIP-seq) will handle this automatically.

Pipeline Tip

Custom Genome Configuration

Reference Genome Selection

Metadata Input *Required

Column Roles

Define Comparisons Multi-group DESeq2

Experimental Design & Metadata

Significance Thresholds

Submission Checklist

Statistical Analysis & Visualization

Submitting Pipeline

Submission Name Required A short, descriptive label for this analysis run. Use something you'll recognise later, e.g. "GSE12345_DrugA_vs_DMSO".

Starting Point Required Choose the file type you are starting from. FASTQ runs the full pipeline (QC → alignment → quantification → statistics). BAM/CRAM skips alignment. Count Matrix skips directly to differential expression.

Assay Type Required Select the sequencing assay used to generate your data. This determines the aligner, reference database, and downstream analysis tools the pipeline will use.

Library Type Required Single-end: One FASTQ file per sample. Paired-end: Two FASTQ files (R1 + R2) per sample. Check your sequencing provider's report if unsure.

Strandedness Unstranded: No strand-specific protocol used. Most common.
fr-firststrand: dUTP/Illumina TruSeq stranded protocol. First read maps to the reverse complement.
fr-secondstrand: Ligation-based. First read maps to the transcript strand. Required by most downstream tools.

FASTQ Files Required Drag and drop gzip-compressed FASTQ files (`.fq.gz` or `.fastq.gz`). For paired-end data, include both R1 and R2 files — they will be matched automatically by filename prefix.

BAM/CRAM Files Required

Count Matrix Required Upload a CSV/TSV with gene IDs in the first column and raw integer counts. Do not upload TPM/FPKM.

Reference Genome Required Select the reference genome matching your organism. The pipeline uses pre-built indices optimized for your specific assay type's aligner.

Metadata Input Required