This document describes the output produced by the pipeline. Most of the plots are taken from the MultiQC report and LncpipeReporter, that summarizes the result at the end of the pipeline.

Pipeline overview

The pipeline is built using Nextflow Plz see a detailed illustration in our literature.

Result Folder structure

Result folder under current path(default) or output_folder set by user. A typical structure of Result is follows:

        ├── QC
        │   ├── N1141_1.clean_fastqc.html
        │   ├── N1141_2.clean_fastqc.html
        │   ├── N1177_1.clean_fastqc.html
        │   └── N1177_2.clean_fastqc.html
        ├── Identified_lncRNA
        │   ├── all_lncRNA_for_classifier.gtf
        │   ├── final_all.fa
        │   ├── final_all.gtf
        │   ├── lncRNA.fa
        │   ├── protein_coding.fa
        │   └── protein_coding.final.gtf
        ├── LncReporter
        │   ├── Differential_Expression_analysis.csv
        │   └── Report.html
        ├── Quantification
        │   ├── kallisto.count.txt
        │   └── kallisto.tpm.txt
        └── Star_alignment
            ├── STAR_N1141
            │   ├── N1141Aligned.sortedByCoord.out.bam
            │   ├── N1141Log.final.out
            │   ├── N1141Log.out
            │   ├── N1141Log.progress.out
            │   └── N1141SJ.out.tab
            └── STAR_N1177
                ├── N1177Aligned.sortedByCoord.out.bam
                ├── N1177Log.final.out
                ├── N1177Log.out
                ├── N1177Log.progress.out
                └── N1177SJ.out.tab
  • QC stored the Quality control output generated by FastQC or AfterQC software.
  • Identified_lncRNA contains all assembled lncRNA and their sequences. all_lncRNA_for_classifier.gtf includes both novel and known lncRNA features in GTF format; lncRNA.fa is all lncRNA sequences in fasta format. protein_coding.final.gtf and protein_coding.fa are protein coding information extracted from gencode annotation. final_all.gtf and final_all.fa are combined files for further analysis.
  • Alignment are hisat/tophat/STAR aligner standard output
  • Quantification are estimated abundance using kallisto. kallisto.count.txt stored reads count matrix and kallisto.tpm.txt are tpm(Transcripts Per Kilobase Million) matrix.
  • LncReporter stored the interactive report file and differential expression matrix generated by LncPipeReporter which wrapped EdgeR.

Output directory: results/QC

  • sample_fastqc.html
    • FastQC report, containing quality metrics for your untrimmed raw fastq files
  • zips/sample_fastqc.zip
    • zip file containing the FastQC report, tab-delimited data file and plot images

Output directory: results/Combined_annotations

  • known.lncRNA.gtf
    • known lncRNA gene annotation file from provided GTF files, can be stored for further comparison
  • gencode_protein_coding.gtf
    • known protein coding gene annotation file from provided GTF files
  • *_mod.gtf (less information )
    • formatted GTF from the provided GTF files by users

Output directory: results/*_alignment

  • *_summary.txt
    • alignment summary files generated by aligner such as tophat, hisat2 and STAR. A typical content are as follow:
      453727 reads; of these:
        453727 (100.00%) were paired; of these:
          434333 (95.73%) aligned concordantly 0 times
          19339 (4.26%) aligned concordantly exactly 1 time
          55 (0.01%) aligned concordantly >1 times
          434333 pairs aligned concordantly 0 times; of these:
            279639 (64.38%) aligned discordantly 1 time
          154694 pairs aligned 0 times concordantly or discordantly; of these:
            309388 mates make up the pairs; of these:
              4095 (1.32%) aligned 0 times
              303301 (98.03%) aligned exactly 1 time
              1992 (0.64%) aligned >1 times
      99.55% overall alignment rate 
  • *sort.bam
    • alignment result in bam format.

Output directory: results/Identified_lncRNA

  • *all_lncRNA_for_classifier.gtf
    • A final identified lncRNA GTF that contains both known lncRNA and novel lncRNAs. It also includes the lncRNA relative position to the nearest genes.
  • final_all.gtf
    • A GTF that contains both lncRNA and protein coding RNA. It includes the lncRNA relative position to the nearest genes.
  • lncRNA.fa
    • All lncRNA sequences in fasta format
  • protein_coding.fa
    • All protein sequences in fasta format
  • final_all.fa
    • All protein + lncRNA sequences in fasta format
  • lncRNA_classification.txt
    • A detailed classification of all lncRNAs according to their coordinate in the chromosome. A detailed illustration of the lncRNA class are shown below
  • lncRNA.mapping.file
    • In the output file, we renamed all lncRNA according their nearest gene for both known and new identified genes. This file contains the name map information between changed name and origin name of the known lncRNAs.

Output directory: results/LncPipeReports

  • LncPipeReporter generated report, plz open the reporter.html directly.

Output directory: results/Merged_assemblies

  • merged.gtf
    • Initial merged GTF by merge assemblies from individual sample. Can be used as input of --merged_gtf parameter

Output directory: results/Quantification

  • kallisto.count.txt
    • reads count matrix at gene level with each line represent a gene and each column represent a sample. NOTE: The second column recorded the gene type, e.g. proten_coding or known lncRNA or novel lncRNA that were not included in the provided gtf
  • kallisto.tpm.txt
    • expression matrix at gene level in TPM format. A detailed explanation of TPM can be found in here