SLAM-seq (Herzog et. al 2017) allows the measurement of gene expression dynamics in the context of total RNA by incorporating 4-thiouridine (4sU) in newly synthesized RNA molecules. Those incorporated 4sU molecules are then converted to Cs giving single nucleotide resolution.
The SLAM-seq pipeline includes Quality Control, rRNA filtering using Bowtie2, Bowtie and STAR prior to Hisat3N alignment for accounting U>C substitutions. The converted and uncorverted reads are generated with a custom Java code, and then gene and isoform expression levels are estimated by RSEM or Kallisto.
- For Quality Control, we use FastQC to create qc outputs. There are optional read quality filtering (trimmomatic), read quality trimming (trimmomatic), adapter removal (cutadapt) processes available.
- Hisat3N accounts for U>C substitutions, which is normally recognized as a mismatch when aligning reads to the genome, and does not penalize when detecting a U>C mismatch on the reads.
- A custom java code is used for generating converted and uncoverted reads where converted reads are of same sets of reads from unconverted reads with U>C substitutions.
- RSEM is used to align converted and unconverted reads to a reference transcripts and estimates gene and isoform expression levels. RSEM incorporates STAR as the main aligner.
- Alternatively, Kallisto used for quantifying abundances of transcripts based on pseudoalignments.
- Genome-wide Bam analysis is done by RseQC, Picard.
- Optionally you can create Integrative Genomics Viewer (IGV) and Genome Browser Files (TDF and Bigwig, respectively)
- Docker: dolphinnext/slamseq_pipeline:1.0
- GitHub: dolphinnext/slamseq_pipeline
- FastQC v0.11.8
- Star v2.6.1
- Hisat2 v2.1.0
- Hisat3N
- OpenJDK v16.0.2
- Picard v2.18.27
- Rseqc v2.6.2
- Samtools v1.3
- Subread v1.6.4
- Multiqc v1.7
- Tophat v2.1.1
- RSEM v1.3.1
- Bowtie2 v2.3.5
- Bowtie v1.2.2
- Trimmomatic v0.39
- Igvtools v2.5.3
- Bedtools v2.27.1
- Fastx_toolkit v0.0.14
- Ucsc-wigToBigWig v366
- Kallisto v0.46.0