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Slam-seq Pipeline

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.

Steps:
  1. 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.
  2. 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.
  3. 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.
  4. 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.
  5. Alternatively, Kallisto used for quantifying abundances of transcripts based on pseudoalignments.
  6. Genome-wide Bam analysis is done by RseQC, Picard.
  7. Optionally you can create Integrative Genomics Viewer (IGV) and Genome Browser Files (TDF and Bigwig, respectively)
Pipeline Container:
  • Docker: dolphinnext/slamseq_pipeline:1.0
  • GitHub: dolphinnext/slamseq_pipeline
Program Versions:
  • 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

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