Sequencing#
RNA#
Library types#
Resume#
Here a figure describing the different RNA-seq library types:
If you don't know the library type of your data you can use GUESSmyLT
Library prep methods#
| kit | Description | Paired | Stranded | Strand according to mRNA | Strand according to first strand |
|---|---|---|---|---|---|
| TruSeq RNA Sample Prep kit | yes | No | fr-unstranded | ||
| SMARTer ultralow RNA protocol | yes | No | fr-unstranded | ||
| All dUTP methods, NSR, NNSR | yes | Yes | RF | fr-firststrand | |
| TruSeq Stranded Total RNA Sample Prep Kit | yes | Yes | RF | fr-firststrand | |
| TruSeq Stranded mRNA Sample Prep Kit | yes | Yes | RF | fr-firststrand | |
| NEB Ultra Directional RNA Library Prep Kit | yes | Yes | RF | fr-firststrand | |
| Agilent SureSelect Strand-Specific | yes | Yes | RF | fr-firststrand | |
| Directional Illumina (Ligation) | yes | Yes | FR | fr-secondstrand | |
| Standard SOLiD | Yes | yes | FR | fr-secondstrand | |
| ScriptSeq v2 RNA-Seq Library Preparation Kit | yes | Yes | FR | fr-secondstrand | |
| SMARTer Stranded Total RNA | yes | Yes | FR | fr-secondstrand | |
| Encore Complete RNA-Seq Library Systems | yes | Yes | FR | fr-secondstrand | |
| NuGEN SoLo | yes | Yes | FR | fr-secondstrand | |
| Illumina ScriptSeq | yes | Yes | FR | fr-secondstrand | |
| SOLiD mate-pair protocol | ff |
--rf orientation are produced using the Illumina mate-pair protocol?
DNA#
Duplicates#
Duplicate reads are defined as originating from a single fragment of RNA/DNA. Duplicates can arise during sample preparation e.g. library construction using PCR (PCR duplication artifacts); single amplification cluster, incorrectly detected as multiple clusters by the optical sensor of the sequencing instrument (optical duplicates). Intereting reading
-
PCR duplicate artifacts Should we remove them. Short answer: NO excepted when using UMIs (e.g. deep sequencing / single cell / etc)
details here -
optical duplicates
Optical, or more broadly Sequencing, duplicates are duplicates that appear clustered together spatially during sequencing and can arise from optical/imagine-processing artifacts or from bio-chemical processes during clonal amplification and sequencing
Tool to perform duplicates analysis (mark, remove): MarkDuplicates (Picard)
Merge read and remove duplicates? See here