{ "id": "epbrby4qew", "slug": "bulk-sequencing", "title": "Bulk sequencing", "updated": "2026-10-25", "topic_path": [ { "id": "life", "label": "Life and health", "api_url": "https://www.edgepedia.org/api/v1/topics/life" }, { "id": "life.biology", "label": "Biological foundations", "api_url": "https://www.edgepedia.org/api/v1/topics/life.biology" }, { "id": "life.biology.genetics", "label": "Genetics and genomic reference", "api_url": "https://www.edgepedia.org/api/v1/topics/life.biology.genetics" }, { "id": "life.biology.genetics.genomics_projects_databases", "label": "Genomics, sequencing, and genome resources", "api_url": "https://www.edgepedia.org/api/v1/topics/life.biology.genetics.genomics_projects_databases" } ], "geo": [ { "id": "geo.nongeo.t1946.life.biology.genetics.genomics_projects_databases", "label": "Non-geographic · 1946 to 2000: Genomics, sequencing, and genome resources", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t1946.life.biology.genetics.genomics_projects_databases", "path": [ { "id": "geo.nongeo", "label": "Non-geographic", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo" }, { "id": "geo.nongeo.t1946", "label": "Non-geographic · 1946 to 2000", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t1946" }, { "id": "geo.nongeo.t1946.life", "label": "Life and health", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t1946.life" }, { "id": "geo.nongeo.t1946.life.biology", "label": "Biological foundations", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t1946.life.biology" }, { "id": "geo.nongeo.t1946.life.biology.genetics", "label": "Genetics and genomic reference", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t1946.life.biology.genetics" }, { "id": "geo.nongeo.t1946.life.biology.genetics.genomics_projects_databases", "label": "Genomics, sequencing, and genome resources", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t1946.life.biology.genetics.genomics_projects_databases" } ] }, { "id": "geo.nongeo.t2001.life.biology.genetics.genomics_projects_databases", "label": "Non-geographic · 2001 to 2020: Genomics, sequencing, and genome resources", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t2001.life.biology.genetics.genomics_projects_databases", "path": [ { "id": "geo.nongeo", "label": "Non-geographic", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo" }, { "id": "geo.nongeo.t2001", "label": "Non-geographic · 2001 to 2020", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t2001" }, { "id": "geo.nongeo.t2001.life", "label": "Life and health", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t2001.life" }, { "id": "geo.nongeo.t2001.life.biology", "label": "Biological foundations", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t2001.life.biology" }, { "id": "geo.nongeo.t2001.life.biology.genetics", "label": "Genetics and genomic reference", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t2001.life.biology.genetics" }, { "id": "geo.nongeo.t2001.life.biology.genetics.genomics_projects_databases", "label": "Genomics, sequencing, and genome resources", "api_url": "https://www.edgepedia.org/api/v1/geo/geo.nongeo.t2001.life.biology.genetics.genomics_projects_databases" } ] } ], "excerpt": "Bulk sequencing is a nucleic acid sequencing approach that reads pooled DNA or RNA from a whole cell population, measuring average expression across hundreds to thousands of cells.", "snippet": "Bulk sequencing is a nucleic acid sequencing approach that reads pooled DNA or RNA from a whole cell population, measuring average expression across hundreds to thousands of cells.", "node": "life.biology.genetics.genomics_projects_databases", "markdown": "# Bulk sequencing\n\nBulk sequencing is a [nucleic acid sequencing](https://www.edgepedia.org/nucleic-acid-sequencing) approach that reads pooled DNA or RNA from a whole cell population or tissue, measuring the average sequence or expression signal across hundreds to thousands of cells rather than resolving individual cells. In a traditional bulk RNA-seq experiment, a tissue sample is ground up and sequenced to measure the average expression level of each gene across all cells in it.[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC11109966/) Bulk RNA-seq is a well-established method that measures average RNA expression in cell populations and is increasingly used in translational and clinical cancer research.[2](https://emea.illumina.com/content/dam/illumina/gcs/assembled-assets/marketing-literature/rna-seq-workflows-guide-m-gl-00034/rna-seq-workflows-guide-m-gl-00034.pdf)\n\n| Key fact | Value |\n|---|---|\n| What it measures | Average gene expression or chromatin state across a whole cell population[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC11109966/) • [3](https://www.science.org/doi/10.1126/science.aab1601) |\n| Typical RNA-seq depth | 10-30 million reads per sample; more than 3.5 million uniquely mapped reads support high-quality human protein-coding analysis[4](https://www.mdpi.com/1422-0067/27/7/3334) |\n| RNA input | 1 ng tolerated (BRB-seq); 10-1000 ng typical for commercial kits[5](https://link.springer.com/article/10.1186/s13059-019-1671-x) • [2](https://emea.illumina.com/content/dam/illumina/gcs/assembled-assets/marketing-literature/rna-seq-workflows-guide-m-gl-00034/rna-seq-workflows-guide-m-gl-00034.pdf) |\n| Cost per sample | Sequencing $10-20; library prep $1.40-$164 depending on protocol[6](https://doi.org/10.1016/j.isci.2026.116984) • [7](https://pmc.ncbi.nlm.nih.gov/articles/PMC10907608/) • [8](https://link.springer.com/article/10.1186/s13059-022-02660-8) |\n| Rare-cell sensitivity | Cell types below ~20% of a heterogeneous sample lack signatures in bulk ATAC-seq[9](https://escholarship.org/content/qt6gc7931k/qt6gc7931k_noSplash_5a6b8862469be4328cbdf58fd82f2b1b.pdf) |\n| Throughput | One NovaSeq X 25B flow cell yields ~520 transcriptomes (≥50M reads each) per run[10](https://supportassets.illumina.com/content/illumina-marketing/amr/en/systems/sequencing-platforms/novaseq-x-plus/specifications.html) |\n\n## How it works\n\nPooling a population changes what the data contain. Each read comes from a random molecule in the pooled extract, so per-gene counts are proportional to the population-average abundance of that transcript, chromatin accessibility, or variant allele. This averaging is the method's core strength and its core weakness: bulk ATAC-seq, like DNase-seq, measures an average of the chromatin states within a population of cells, masking heterogeneity between and within cell types.[3](https://www.science.org/doi/10.1126/science.aab1601) A signal from a rare subpopulation is diluted by the majority; in heterogeneous tissue, bulk ATAC-seq profiles lack signatures of rare cell types below about 20% of total cells.[9](https://escholarship.org/content/qt6gc7931k/qt6gc7931k_noSplash_5a6b8862469be4328cbdf58fd82f2b1b.pdf) Bulk RNA-seq data are also a snapshot in time, so studying a biological process requires samples collected at multiple timepoints.[11](https://www.montana.edu/mbi/facilities/cellular-analysis-core/additional-resources/documents/Bulk-RNAseq-Instructional-Manual.pdf)\n\n## How it is done\n\n**Bulk DNA sequencing** (whole-genome) involves isolating genomic DNA, shearing it to fragments of approximately 100-500 base pairs, end repair, adapter ligation, PCR amplification, sequencing, and alignment to a reference genome for variant calling.[12](https://pmc.ncbi.nlm.nih.gov/articles/PMC6741438/)\n\n**Bulk RNA-seq** follows a parallel path. A standard NEBNext Ultra II-style protocol starts with mRNA enrichment or rRNA depletion, fragments the RNA, reverse transcribes it to cDNA, and ligates adapters; indexed libraries are then generated by PCR enrichment with unique index primers, and libraries are pooled on Illumina flow cells using unique 5' and 3' index primers per sample.[11](https://www.montana.edu/mbi/facilities/cellular-analysis-core/additional-resources/documents/Bulk-RNAseq-Instructional-Manual.pdf)\n\n**Analysis** is standardized. Raw BCL files are converted to FastQ and quality-assessed with FastQC; quantification then follows one of two routes: reads are aligned with STAR and counted per gene with featureCounts, or transcripts are quantified directly with Salmon pseudoalignment before normalization.[11](https://www.montana.edu/mbi/facilities/cellular-analysis-core/additional-resources/documents/Bulk-RNAseq-Instructional-Manual.pdf) For DNA, a Snakemake/GATK pipeline using Bowtie2 alignment and GATK HaplotypeCaller can identify useful variants at average coverage as low as 10x.[12](https://pmc.ncbi.nlm.nih.gov/articles/PMC6741438/)\n\n## Origin\n\nTwo 1977 papers opened [DNA sequencing](https://www.edgepedia.org/dna-sequencing). F. Sanger, S. Nicklen, and A. R. Coulson introduced the chain-terminator method using dideoxynucleoside triphosphates as chain-terminating inhibitors of [DNA polymerase](https://www.edgepedia.org/dna-polymerase)[13](https://doi.org/10.1073/pnas.74.12.5463), and A M Maxam and W Gilbert introduced a chemical procedure that breaks a terminally labeled DNA molecule partially at each repetition of a base.[14](https://doi.org/10.1073/pnas.74.2.560) [Shotgun sequencing](https://www.edgepedia.org/shotgun-sequencing), automated fluorescence Sanger machines, and the 2005 454 pyrosequencing platform by Marcel Margulies and colleagues[15](https://doi.org/10.1038/nature03959) set the stage for high-throughput practice.\n\nFor transcriptomics, Victor E. Velculescu, Lin Zhang, Bert Vogelstein, and [Kenneth W. Kinzler](https://www.edgepedia.org/kenneth-w-kinzler) introduced SAGE in 1995[16](https://doi.org/10.1126/science.270.5235.484) and [Sydney Brenner](https://www.edgepedia.org/sydney-brenner) and colleagues introduced massively parallel signature sequencing in 2000[17](https://doi.org/10.1038/76469) as forerunners. RNA-seq itself was developed by five groups in 2008[18](https://arep.med.harvard.edu/pdf/Shendure_Waterston_2017.pdf); among them, Ali Mortazavi and colleagues published the mammalian RNA-Seq paper in [Nature Methods](https://www.edgepedia.org/nature-methods)[19](https://doi.org/10.1038/nmeth.1226), and Ugrappa Nagalakshmi and colleagues published the yeast transcriptome paper in Science.[20](https://doi.org/10.1126/science.1158441)\n\n## Variants\n\n**Bulk RNA-seq** splits by RNA selection: mRNA-seq selects polyA-tailed protein-coding transcripts before library prep, while total RNA-seq uses rRNA depletion (for example Ribo-Zero Plus) to capture coding and noncoding transcripts, including from FFPE or degraded samples.[2](https://emea.illumina.com/content/dam/illumina/gcs/assembled-assets/marketing-literature/rna-seq-workflows-guide-m-gl-00034/rna-seq-workflows-guide-m-gl-00034.pdf) Library protocols divide into late-multiplexing strand kits (TruSeq, NEBNext) and 3'-end early-barcoding tag protocols. BRB-seq introduces a sample-specific barcode during reverse transcription so samples are pooled before all later steps[5](https://link.springer.com/article/10.1186/s13059-019-1671-x); prime-seq, based on SCRB-seq and mcSCRB-seq, uses poly(A) priming, template switching, early barcoding, and UMIs.[8](https://link.springer.com/article/10.1186/s13059-022-02660-8)\n\n**Bulk ATAC-seq** profiles open chromatin by Tn5 transposition of native chromatin, introduced by Jason D Buenrostro, Paul G Giresi, Lisa C Zaba, Howard Y Chang, and William J Greenleaf in 2013[21](https://doi.org/10.1038/nmeth.2688), building on earlier Tn5 in vitro transposition work by Igor Yu Goryshin and [William S. Reznikoff](https://www.edgepedia.org/william-s-reznikoff)[22](https://doi.org/10.1074/jbc.273.13.7367) and high-density in vitro transposition library construction by Andrew Adey and colleagues.[23](https://doi.org/10.1186/gb-2010-11-12-r119) It needs fewer than 50,000 cells and no prior knowledge of epigenetic marks[9](https://escholarship.org/content/qt6gc7931k/qt6gc7931k_noSplash_5a6b8862469be4328cbdf58fd82f2b1b.pdf), and streamlined versions enable population epigenotyping on single small individuals such as [Daphnia pulex](https://www.edgepedia.org/daphnia-pulex) and adult [Schistosoma mansoni](https://www.edgepedia.org/schistosoma-mansoni) worms (~10,000 cells).[24](https://wellcomeopenresearch.org/articles/5-121)\n\n## Applications\n\nRecommended minimum read depth for gene expression comparisons is at least 8 million reads for bacteria, 20 million for mammals, and 40 million for plants[11](https://www.montana.edu/mbi/facilities/cellular-analysis-core/additional-resources/documents/Bulk-RNAseq-Instructional-Manual.pdf); 10-30 million reads per sample is the typical range, and more than 3.5 million uniquely mapped reads already support high-quality human protein-coding analysis.[4](https://www.mdpi.com/1422-0067/27/7/3334) Illumina library prep inputs span 25-1000 ng high-quality RNA for polyA-selection ligation-based prep, about 10 ng for fragmentation-based prep, and 20 ng FFPE RNA for hybrid-capture workflows[2](https://emea.illumina.com/content/dam/illumina/gcs/assembled-assets/marketing-literature/rna-seq-workflows-guide-m-gl-00034/rna-seq-workflows-guide-m-gl-00034.pdf); BRB-seq tolerates as little as 1 ng.[5](https://link.springer.com/article/10.1186/s13059-019-1671-x)\n\nLibrary prep, not sequencing, now dominates cost. Sequencing an RNA-seq sample costs $10-20, with one million reads under one dollar on NovaSeq X machines, while commercial library kits cost $60-164 per sample.[6](https://doi.org/10.1016/j.isci.2026.116984) Low-cost tag protocols cut this sharply: prime-seq reagents cost $2.53 per sample in 96-sample batches versus $60 (NEBNext) to $164 (SMARTer Stranded) for commercial kits[8](https://link.springer.com/article/10.1186/s13059-022-02660-8), and BOLT-seq constructs 3'-end libraries from unpurified lysates in under one day at $1.40 per sample versus $45-47 with NEBNext Ultra II.[7](https://pmc.ncbi.nlm.nih.gov/articles/PMC10907608/) One NovaSeq X 25B flow cell yields about 520 transcriptomes of at least 50 million reads per run.[10](https://supportassets.illumina.com/content/illumina-marketing/amr/en/systems/sequencing-platforms/novaseq-x-plus/specifications.html) Published sources do not quantify current bulk whole-genome or whole-exome depth and cost.\n\n## Limitations and alternatives\n\n**Averaging and lost cell-type information.** Bulk data mask rare cell populations and dynamic processes such as differentiation, immune activation, and epithelial-mesenchymal transition[4](https://www.mdpi.com/1422-0067/27/7/3334), and heterogeneous samples lack signatures of cell types below ~20%.[9](https://escholarship.org/content/qt6gc7931k/qt6gc7931k_noSplash_5a6b8862469be4328cbdf58fd82f2b1b.pdf) For rare clones, the best-quantified design is bulk segregant analysis, which pools ~50 mutant progeny so causal variants appear at high frequency while non-causal variants segregate at ~50%; a non-causal variant reaches allele frequency ≥0.67 in the mutant pool with 5% probability.[12](https://pmc.ncbi.nlm.nih.gov/articles/PMC6741438/) Published sources do not state a general variant allele fraction at which rare-clone detection fails.\n\n**Protocol limits.** 3'-end early-multiplexing methods (CEL-seq2, SCRB-seq, STRT-seq derivatives) cannot address splicing, fusion genes, or [RNA editing](https://www.edgepedia.org/rna-editing) because reads cover only transcript ends.[5](https://link.springer.com/article/10.1186/s13059-019-1671-x) ATAC-seq carries Tn5 sequence-insertion biases and is not well suited to per-locus transcription factor footprinting.[9](https://escholarship.org/content/qt6gc7931k/qt6gc7931k_noSplash_5a6b8862469be4328cbdf58fd82f2b1b.pdf)\n\n**Deconvolution as a partial remedy.** The core model is \\( b = S \\cdot p \\), where \\( b \\) is the bulk expression vector of \\( n \\) genes, \\( S \\) an \\( n \\)-genes-by-\\( k \\)-cell-types signature matrix, and \\( p \\) the cell-type proportion vector summing to one.[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC11109966/) [Performance](https://www.edgepedia.org/performance) declines substantially in cross-reference settings where bulk data and single-cell references come from different donors, studies, batches, or platforms, and differences in cell-type-specific mRNA content can systematically distort estimated cell fractions.[4](https://www.mdpi.com/1422-0067/27/7/3334) On the analysis side, BLUE, a U-Net-based deep learning algorithm, deconvolves bulk RNA-seq into cell-type proportions and cell-type-specific expression profiles, and applied to TCGA AML bulk RNA-seq identified three patient subtypes with distinctive survival outcomes validated in the independent TARGET cohort.[25](https://journals.plos.org/ploscompbiol/article?id=10.1371%2Fjournal.pcbi.1014101)\n\n## References\n\n1. [Fourteen years of cellular deconvolution: methodology, applications, technical evaluation and outstanding challenges (Nucleic Acids Research, 2024)](https://pmc.ncbi.nlm.nih.gov/articles/PMC11109966/)\n2. [RNA-Seq Workflows Guide (Illumina)](https://emea.illumina.com/content/dam/illumina/gcs/assembled-assets/marketing-literature/rna-seq-workflows-guide-m-gl-00034/rna-seq-workflows-guide-m-gl-00034.pdf)\n3. [Multiplex single-cell profiling of chromatin accessibility by combinatorial cellular indexing (Cusanovich et al., Science 2015)](https://www.science.org/doi/10.1126/science.aab1601)\n4. [Integration of Bulk and Single-Cell RNA Sequencing Analyses in Biomedicine (International Journal of Molecular Sciences, 2025)](https://www.mdpi.com/1422-0067/27/7/3334)\n5. [BRB-seq: ultra-affordable high-throughput transcriptomics enabled by bulk RNA barcoding and sequencing (Aliee et al., Genome Biology 2019)](https://link.springer.com/article/10.1186/s13059-019-1671-x)\n6. [Increasing usable reads in RNA-seq protocols (iScience, 2026)](https://doi.org/10.1016/j.isci.2026.116984)\n7. [BOLT-seq: cost and time-efficient construction of a 3'-end mRNA library from unpurified bulk RNA in a single tube (2024)](https://pmc.ncbi.nlm.nih.gov/articles/PMC10907608/)\n8. [Prime-seq, efficient and powerful bulk RNA sequencing (Janjic et al., Genome Biology 2022)](https://link.springer.com/article/10.1186/s13059-022-02660-8)\n9. [Chromatin accessibility profiling by ATAC-seq (Omni-ATAC Nature Protocols protocol, repository copy)](https://escholarship.org/content/qt6gc7931k/qt6gc7931k_noSplash_5a6b8862469be4328cbdf58fd82f2b1b.pdf)\n10. [NovaSeq X Series specifications (Illumina)](https://supportassets.illumina.com/content/illumina-marketing/amr/en/systems/sequencing-platforms/novaseq-x-plus/specifications.html)\n11. [Bulk RNA-seq Instructional Manual (Montana State University Cellular Analysis Core)](https://www.montana.edu/mbi/facilities/cellular-analysis-core/additional-resources/documents/Bulk-RNAseq-Instructional-Manual.pdf)\n12. [Whole genome sequencing of yeast cells (Current Protocols)](https://pmc.ncbi.nlm.nih.gov/articles/PMC6741438/)\n13. [F. Sanger, S. Nicklen, A. R. Coulson (1977). DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences.](https://doi.org/10.1073/pnas.74.12.5463)\n14. [A M Maxam, W Gilbert (1977). A new method for sequencing DNA.. Proceedings of the National Academy of Sciences.](https://doi.org/10.1073/pnas.74.2.560)\n15. [Marcel Margulies and colleagues (2005). Genome sequencing in microfabricated high-density picolitre reactors. Nature.](https://doi.org/10.1038/nature03959)\n16. [Victor E. Velculescu and colleagues (1995). Serial Analysis of Gene Expression. Science.](https://doi.org/10.1126/science.270.5235.484)\n17. [Sydney Brenner and colleagues (2000). Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays. Nature Biotechnology.](https://doi.org/10.1038/76469)\n18. [DNA sequencing at 40: past, present and future (Shendure & Waterston, 2017)](https://arep.med.harvard.edu/pdf/Shendure_Waterston_2017.pdf)\n19. [Ali Mortazavi and colleagues (2008). Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nature Methods.](https://doi.org/10.1038/nmeth.1226)\n20. [Ugrappa Nagalakshmi and colleagues (2008). The Transcriptional Landscape of the Yeast Genome Defined by RNA Sequencing. Science.](https://doi.org/10.1126/science.1158441)\n21. [Jason D Buenrostro and colleagues (2013). Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nature Methods.](https://doi.org/10.1038/nmeth.2688)\n22. [Igor Yu Goryshin, William S. Reznikoff (1998). Tn5 in Vitro Transposition. Journal of Biological Chemistry.](https://doi.org/10.1074/jbc.273.13.7367)\n23. [Andrew Adey and colleagues (2010). Rapid, low-input, low-bias construction of shotgun fragment libraries by high-density in vitro transposition. Genome biology.](https://doi.org/10.1186/gb-2010-11-12-r119)\n24. [A simple ATAC-seq protocol for population epigenomics (Wellcome Open Research)](https://wellcomeopenresearch.org/articles/5-121)\n25. [Deconvolving cell-type-specific gene expression profiles from bulk RNA-seq samples (BLUE, PLOS Computational Biology)](https://journals.plos.org/ploscompbiol/article?id=10.1371%2Fjournal.pcbi.1014101)\n\n---\n*Topic: Encyclopedia › Life and health › Biological foundations › Genetics and genomic reference › Genomics, sequencing, and genome resources*\n\n*Initially written Sep 29, 2026 · Edited: Oct 15, 2026; Oct 25, 2026*\n\n*Copyright 2026 EdgeChat AI, a subsidiary of Biostate AI.*\n\nLicense: Edgepedia Community License 1.0, https://www.edgepedia.org/edgepedia/license\n", "same_as": [], "url": "https://www.edgepedia.org/bulk-sequencing", "markdown_url": "https://www.edgepedia.org/bulk-sequencing.md", "license": { "name": "Edgepedia Community License 1.0", "url": "https://www.edgepedia.org/edgepedia/license", "summary": "Free with credit, commercial use included. AI training is open to everyone. For other uses, organizations over USD 100M in revenue or 100M monthly users license separately.", "spdx": "LicenseRef-Edgepedia-Community-1.0" }, "credit": "\"Bulk sequencing\", Edgepedia (EdgeChat), https://www.edgepedia.org/bulk-sequencing. Edgepedia Community License 1.0.", "credit_md": "\"[Bulk sequencing](https://www.edgepedia.org/bulk-sequencing)\", Edgepedia (EdgeChat), [https://www.edgepedia.org/bulk-sequencing](https://www.edgepedia.org/bulk-sequencing). [Edgepedia Community License 1.0](https://www.edgepedia.org/edgepedia/license).", "credit_html": "\"Bulk sequencing\", Edgepedia (EdgeChat), https://www.edgepedia.org/bulk-sequencing. Edgepedia Community License 1.0.", "speakable": "Bulk sequencing is a nucleic acid sequencing approach that reads pooled DNA or RNA from a whole cell population, measuring average expression across hundreds to thousands of cells." }