a b/docs/mapping_small_rna.md 

  1
  
# Mapping small RNA-seq





  
  

  2
  





  
  

  3
  
## Prepare genome annotation





  
  

  4
  





  
  

  5
  
For mapping of small RNA-seq reads, exSEEK adopts sequential mapping strategy, which assign reads to gene annotations sequentially according the the ordered defined by the user.





  
  

  6
  
By default, exSEEK assign reads in the following order:





  
  

  7
  





  
  

  8
  
spike-in, rRNA, lncRNA, miRNA, mRNA, piRNA, snoRNA, snRNA, srpRNA, tRNA, tucpRNA, Y_RNA, genome, circRNA





  
  

  9
  





  
  

  10
  
We derived the genome annotation file from various sources: 





  
  

  11
  





  
  

  12
  
| Type | Number of genes | Source |





  
  

  13
  
| :--- | :--- | :--- |





  
  

  14
  
| miRNA | 1917 | miRBase hairpin \(Version 22\) |





  
  

  15
  
| piRNA | 23431 | piRNABank |





  
  

  16
  
| lncRNA | 15778 | GENCODE V27 and mitranscriptome |





  
  

  17
  
| rRNA | 37 | NCBI refSeq 109 |





  
  

  18
  
| mRNA | 19836 | GENCODE V27 |





  
  

  19
  
| snoRNA | 943 | GENCODE V27 |





  
  

  20
  
| snRNA | 1900 | GENCODE V27 |





  
  

  21
  
| srpRNA | 680 | GENCODE V27 |





  
  

  22
  
| tRNA | 649 | GENCODE V27 |





  
  

  23
  
| tucpRNA | 3734 | GENCODE V27 |





  
  

  24
  
| Y\_RNA | 756 | GENCODE V27 |





  
  

  25
  
| circRNA | 140527 | circBase |





  
  

  26
  
| repeats | - | UCSC Genome Browser \(rmsk\) |





  
  

  27
  
| promoter | - | ChromHMM tracks from 9 cell lines from UCSC Genome Browser |





  
  

  28
  
| enhancer | - | ChromHMM tracks from 9 cell lines from UCSC Genome Browser |





  
  

  29
  





  
  

  30
  
spike-in is a special type of genome annotation that should be provided by the user if spike-in sequences are used. 





  
  

  31
  





  
  

  32
  
The paths of the bowtie2 index files:





  
  

  33
  





  
  

  34
  
| Type | FASTA file | bowtie2 index file |





  
  

  35
  
| :--- | :--- | :--- |





  
  

  36
  
| spike-in | `${genome_dir}/fasta/spikein_small.fa` | `${genome_dir}/index/bowtie2/spikein` |





  
  

  37
  
| rRNA | `${genome_dir}/fasta/rRNA.fa` | `${genome_dir}/index/bowtie2/rRNA` |





  
  

  38
  
| miRNA | `${genome_dir}/fasta/miRNA.fa` | `${genome_dir}/rsem_index/bowtie2/miRNA` |





  
  

  39
  
| piRNA | `${genome_dir}/fasta/piRNA.fa` | `${genome_dir}/rsem_index/bowtie2/piRNA` |





  
  

  40
  
| lncRNA | `${genome_dir}/fasta/lncRNA.fa` | `${genome_dir}/rsem_index/bowtie2/lncRNA` |





  
  

  41
  
| mRNA | `${genome_dir}/fasta/mRNA.fa` | `${genome_dir}/rsem_index/bowtie2/mRNA` |





  
  

  42
  
| snoRNA | `${genome_dir}/fasta/snoRNA.fa` | `${genome_dir}/rsem_index/bowtie2/snoRNA` |





  
  

  43
  
| snRNA | `${genome_dir}/fasta/snRNA.fa` | `${genome_dir}/rsem_index/bowtie2/snRNA` |





  
  

  44
  
| srpRNA | `${genome_dir}/fasta/srpRNA.fa` | `${genome_dir}/rsem_index/bowtie2/srpRNA` |





  
  

  45
  
| tRNA | `${genome_dir}/fasta/tRNA.fa` | `${genome_dir}/rsem_index/bowtie2/tRNA` |





  
  

  46
  
| tucpRNA | `${genome_dir}/fasta/tucpRNA.fa` | `${genome_dir}/rsem_index/bowtie2/tucpRNA` |





  
  

  47
  
| Y_RNA | `${genome_dir}/fasta/Y_RNA.fa` | `${genome_dir}/rsem_index/bowtie2/Y_RNA` |





  
  

  48
  
| circRNA | `${genome_dir}/fasta/circRNA.fa` | `${genome_dir}/rsem_index/bowtie2/circRNA` |





  
  

  49
  





  
  

  50
  
**Note**: `${genome_dir}` is the root directory of genome annotation files.





  
  

  51
  





  
  

  52
  
### Build bowtie2 index for spike-in sequences





  
  

  53
  





  
  

  54
  
If your samples contain spike-in sequences, you should first prepare a FASTA file of your spike-in sequences and copy it to `${genome_dir}/fasta/spikein_small.fa`. 





  
  

  55
  
Then create an index file (`${genome_dir}/fasta/spikein_small.fai`) by the following command:





  
  

  56
  





  
  

  57
  
```bash





  
  

  58
  
samtools faidx ${genome_dir}/fasta/spikein_small.fa





  
  

  59
  
```





  
  

  60
  





  
  

  61
  
Run the following commands to build bowtie2 index files for spike-in sequences:





  
  

  62
  





  
  

  63
  
```bash





  
  

  64
  
cut -f1,2 ${genome_dir}/fasta/spikein_small.fa.fai > ${genome_dir}/chrom_sizes/spikein_small





  
  

  65
  
{





  
  

  66
  
    echo -e 'chrom\tstart\tend\tname\tscore\tstrand\tgene_id\ttranscript_id\tgene_name\ttranscript_name\tgene_type\ttranscript_type\tsource'





  
  

  67
  
    awk 'BEGIN{OFS="\t";FS="\t"}{print $1,0,$2,$1,0,"+",$1,$1,$1,$1,"spikein","spikein","spikein"}' ${genome_dir}/fasta/spikein_small.fa.fai





  
  

  68
  
} > ${genome_dir}/transcript_table/spikein_small.txt





  
  

  69
  
bowtie2-build ${genome_dir}/fasta/spikein_small.fa ${genome_dir}/index/bowtie2/spikein_small





  
  

  70
  
```