---
description: Analyzing RNA-seq data with Salmon/STAR/RSEM
---

# Mapping & quantification

## Strategies

| Methods         | Tools                                                                                                                                                     | Output Quantification                                                                                | Total Time (hr)                                                  |
| --------------- | --------------------------------------------------------------------------------------------------------------------------------------------------------- | ---------------------------------------------------------------------------------------------------- | ---------------------------------------------------------------- |
| Alignment-free  | [Salmon](alignment-free-method#salmon-mapping-based-mode)                                                                                                 | <p><strong>Transcript-level</strong> </p><p>(gene-level aggregation using <code>tximport</code>)</p> | **3.4**                                                          |
| Alignment-free  | <p><a href="alignment-free-method#salmon-with-bootstrap">Salmon <br>+bootstraps</a></p>                                                                   | <p><strong>Transcript-level</strong> </p><p>(gene-level aggregation using <code>tximport</code>)</p> | **16.0**                                                         |
| Alignment-free  | <p><a href="alignment-free-method#kallisto-with-bootstrap">Kallisto</a></p><p><a href="alignment-free-method#kallisto-with-bootstrap">+bootstraps</a></p> | <p><strong>Transcript-level</strong> </p><p>(gene-level aggregation using <code>tximport</code>)</p> | **6.1**                                                          |
| Alignment-based | [STAR](alignment-based-method#star-alignment)                                                                                                             | <p><strong>Gene-level</strong></p><p>(with <code>--quantMode GeneCounts</code> option)</p>           | **14.8**                                                         |
| Alignment-based | [STAR+Salmon](alignment-based-method#star-salmon-quantification-alignment-based-mode)                                                                     | <p><strong>Transcript-level</strong> </p><p>(gene-level aggregation using <code>tximport</code>)</p> | <p><strong>16.3</strong> </p><p>STAR: 14.8</p><p>Salmon: 1.5</p> |
| Alignment-based | [STAR+RSEM](alignment-based-method#star-rsem-quantification)                                                                                              | **Gene-level** and **Transcript-level**                                                              | <p><strong>57.2</strong> </p><p>STAR: 14.8</p><p>RSEM: 42.3</p>  |

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CPU usage is limited to 6 threads and memory to 32 GB where applicable
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## LibType and strandedness

|       Tools      | dUTP, NSR, NNSR                                                       | Ligation, Standard SOLiD                                              | Standard Illumina                         |
| :--------------: | --------------------------------------------------------------------- | --------------------------------------------------------------------- | ----------------------------------------- |
| TopHat/Cufflinks | fr-firststrand                                                        | fr-secondstrand                                                       | fr-unstranded                             |
|      HISAT2      | <p>--rna-strandness RF (Paired)</p><p>--rna-strandness R (Single)</p> | <p>--rna-strandness FR (Paired)</p><p>--rna-strandness F (Single)</p> |                                           |
|      Salmon      | <p>-l ISR (Paired)</p><p>-l SR (Single)</p>                           | <p>-l ISF (Paired)</p><p>-l SF (Single)</p>                           | <p>-l IU (Paired)</p><p>-l U (Single)</p> |
|     Kallisto     | --rf-stranded                                                         | --fr-stranded                                                         |                                           |
|       RSEM       | --strandedness reverse                                                | --strandedness forward                                                | --strandedness none                       |
|   FeatureCounts  | -s 2                                                                  | -s 1                                                                  | -s 0                                      |
|       HTSeq      | --stranded reverse                                                    | --stranded yes                                                        | --stranded no                             |
