Small RNA sequencing processing in the example of smallRNA_7

1. adapter sequence

   Lexogen small RNA-Seq kit
   
   some common adapter sequences from different kits for reference:
   
       - TruSeq Small RNA (Illumina): TGGAATTCTCGGGTGCCAAGG
       - Small RNA Kits V1 (Illumina): TCGTATGCCGTCTTCTGCTTGT
       - Small RNA Kits V1.5 (Illumina): ATCTCGTATGCCGTCTTCTGCTTG
       - NEXTflex Small RNA Sequencing Kit v3 for Illumina Platforms (Bioo Scientific): TGGAATTCTCGGGTGCCAAGG
       - LEXOGEN Small RNA-Seq Library Prep Kit (Illumina): TGGAATTCTCGGGTGCCAAGGAACTCCAGTCAC
   
   [Header],,,,,
   IEMFileVersion,4,,,,
   InvestigatorName,ag96,,,,
   ExperimentName,ag96,,,,
   Date,16.10.2023,,,,
   Workflow,GenerateFASTQ,,,,
   Application,NextSeqFASTQOnly,,,,
   Assay,TruSeq HT,,,,
   Description,pcr,,,,
   Chemistry,Amplicon,,,,
   ,,,,,
   [Reads],,,,,
   82,,,,,
   ,,,,,
   ,,,,,
   [Settings],,,,,
   Adapter,TGGAATTCTCGGGTGCCAAGGAACTCCAGTCAC,,,,
   ,,,,,
   ,,,,,
   [Data],,,,,
   Sample_ID,Sample_Name,I7_Index_ID,index,Sample_Project,Description
   nf930,01_0505_WaGa_wt_EV_RNA,SRi7001,CAGCGT,2023_064_nf_ute,smallRNA-Seq
   nf931,02_0505_WaGa_sT_DMSO_EV_RNA,SRi7002,GATCAC,2023_064_nf_ute,smallRNA-Seq
   nf932,03_0505_WaGa_sT_Dox_EV_RNA,SRi7003,ACCAGT,2023_064_nf_ute,smallRNA-Seq
   nf933,04_0505_WaGa_scr_DMSO_EV_RNA,SRi7004,TGCACG,2023_064_nf_ute,smallRNA-Seq
   nf934,05_0505_WaGa_scr_Dox_EV_RNA,SRi7005,ACATTA,2023_064_nf_ute,smallRNA-Seq
   nf935,06_1905_WaGa_wt_EV_RNA,SRi7006,GTGTAG,2023_064_nf_ute,smallRNA-Seq
   nf936,07_1905_WaGa_sT_DMSO_EV_RNA,SRi7007,CTAGTC,2023_064_nf_ute,smallRNA-Seq
   nf937,08_1905_WaGa_sT_Dox_EV_RNA,SRi7008,TGTGCA,2023_064_nf_ute,smallRNA-Seq
   nf938,09_1905_WaGa_scr_DMSO_EV_RNA,SRi7009,TCAGGA,2023_064_nf_ute,smallRNA-Seq
   nf939,10_1905_WaGa_scr_Dox_EV_RNA,SRi7010,CGGTTA,2023_064_nf_ute,smallRNA-Seq
   nf940,11_control_MKL1,SRi7011,TTAACT,2023_064_nf_ute,smallRNA-Seq
   nf941,12_control_WaGa,SRi7012,ATGAAC,2023_064_nf_ute,smallRNA-Seq
   

2. input data

   ln -s ./231016_NB501882_0435_AHG7HMBGXV/nf930/01_0505_WaGa_wt_EV_RNA_S1_R1_001.fastq.gz         0505_WaGa_wt.fastq.gz
   ln -s ./231016_NB501882_0435_AHG7HMBGXV/nf931/02_0505_WaGa_sT_DMSO_EV_RNA_S2_R1_001.fastq.gz    0505_WaGa_sT_DMSO.fastq.gz
   ln -s ./231016_NB501882_0435_AHG7HMBGXV/nf932/03_0505_WaGa_sT_Dox_EV_RNA_S3_R1_001.fastq.gz     0505_WaGa_sT_Dox.fastq.gz
   ln -s ./231016_NB501882_0435_AHG7HMBGXV/nf933/04_0505_WaGa_scr_DMSO_EV_RNA_S4_R1_001.fastq.gz   0505_WaGa_scr_DMSO.fastq.gz
   ln -s ./231016_NB501882_0435_AHG7HMBGXV/nf934/05_0505_WaGa_scr_Dox_EV_RNA_S5_R1_001.fastq.gz    0505_WaGa_scr_Dox.fastq.gz
   ln -s ./231016_NB501882_0435_AHG7HMBGXV/nf935/06_1905_WaGa_wt_EV_RNA_S6_R1_001.fastq.gz         1905_WaGa_wt.fastq.gz
   ln -s ./231016_NB501882_0435_AHG7HMBGXV/nf936/07_1905_WaGa_sT_DMSO_EV_RNA_S7_R1_001.fastq.gz    1905_WaGa_sT_DMSO.fastq.gz
   ln -s ./231016_NB501882_0435_AHG7HMBGXV/nf937/08_1905_WaGa_sT_Dox_EV_RNA_S8_R1_001.fastq.gz     1905_WaGa_sT_Dox.fastq.gz
   ln -s ./231016_NB501882_0435_AHG7HMBGXV/nf938/09_1905_WaGa_scr_DMSO_EV_RNA_S9_R1_001.fastq.gz   1905_WaGa_scr_DMSO.fastq.gz
   ln -s ./231016_NB501882_0435_AHG7HMBGXV/nf939/10_1905_WaGa_scr_Dox_EV_RNA_S10_R1_001.fastq.gz   1905_WaGa_scr_Dox.fastq.gz
   ln -s ./231016_NB501882_0435_AHG7HMBGXV/nf940/11_control_MKL1_S11_R1_001.fastq.gz               control_MKL1.fastq.gz
   ln -s ./231016_NB501882_0435_AHG7HMBGXV/nf941/12_control_WaGa_S12_R1_001.fastq.gz               control_WaGa.fastq.gz
   

3. run cutadapt

   for sample in 0505_WaGa_wt 0505_WaGa_sT_DMSO 0505_WaGa_sT_Dox 0505_WaGa_scr_DMSO 0505_WaGa_scr_Dox 1905_WaGa_wt 1905_WaGa_sT_DMSO 1905_WaGa_sT_Dox 1905_WaGa_scr_DMSO 1905_WaGa_scr_Dox  control_MKL1 control_WaGa; do
     cutadapt -a TGGAATTCTCGGGTGCCAAGGAACTCCAGTCAC -q 20 -o ${sample}2.fastq.gz --minimum-length 5 --trim-n ${sample}.fastq.gz >> LOG
   done
   #jhuang@hamburg:~/DATA/Data_Ute/Data_Ute_smallRNA_7$ fastp -i 0505_WaGa_wt.fastq.gz -o 0505_WaGa_wt3.fastq.gz -a TGGAATTCTCGGGTGCCAAGGAACTCCAGTCAC
   

4. run COMPSRA

   ln -s ../Data_Ute_smallRNA_3/bundle_v1 .
   
   # DEBUG_1: Make sure the file COMPSRA.jar under Data_Ute_smallRNA_7
   # DEBUG_2: "-qc -ra TGGAATTCTCGGGTGCCAAGGAACTCCAGTCAC -rb 4" does not work! Using cutadapt -a xxxx -q 20 replace those parameters!
   for sample in 0505_WaGa_wt 0505_WaGa_sT_DMSO 0505_WaGa_sT_Dox 0505_WaGa_scr_DMSO 0505_WaGa_scr_Dox 1905_WaGa_wt 1905_WaGa_sT_DMSO 1905_WaGa_sT_Dox 1905_WaGa_scr_DMSO 1905_WaGa_scr_Dox  control_MKL1 control_WaGa; do
     mkdir our_out/${sample}2/
     java -jar COMPSRA.jar -ref hg38       -rh 20 -rt 20 -rr 20 -rlh 8,17 -aln -mt star -ann -ac 1,2,3,4,5,6  -in ${sample}2.fastq.gz -out ./our_out/
   done
   
   #4.2.3 -rb/-rm_bias n
   #To remove n random bases in both 5’ (5-prime) and 3’ (3-prime) ends after removing the adapter sequence.
   #4.2.4 -rh/-rm_low_quality_head score
   #To remove the low quality bases with the score less than score from 5’ (5-prime) end.
   #4.2.5 -rt/-rm_low_quality_tail score
   #To remove the low quality bases with the score less than score from 3’ (3-prime) end.
   #4.2.6 -rr/-rm_low_quality_read score
   #To remove the low quality reads with the average score less than score.
   #4.6.3 -fdclass/-fun_diff_class A1,A2,...,An
   #To set the small RNAs that will be performed the differential expression analysis. The format is the same as the parameter -ac/-ann_class A1,A2,...,An.
   #4.6.4 -fdcase/-fun_diff_case ID1,ID2,...,IDn
   #To set the IDs of case samples.
   #4.6.5 -fdctrl/-fun_diff_control ID1,ID2,...,IDn
   #To set the IDs of control samples.
   #4.4.2 -ac/-ann_class A1,A2,...,An
   #To set the small RNA categories that will be annotated. The index of small RNA is listed:
   #    1 miRNA
   #    2 piRNA
   #    3 tRNA
   #    4 snoRNA
   #    5 snRNA
   #    6 circRNA
   
   java -jar COMPSRA.jar -ref hg38 -fun -fm -fms 1-5 -fdclass 1,2,3,4,5 -fdann -pro COMPSRA_MERGE -inf ./sample.list -out ./our_out/
   java -jar COMPSRA.jar -ref hg38 -fun -fd -fdclass 1,2,3,4,5 -fdcase 1-2 -fdctrl 3-6 -fdnorm cpm -fdtest mwu -fdann -pro COMPSRA_DEG -inf ./sample.list -out ./our_out/
   

5. The results without using cutadapt for comparison

   mkdir our_out
   for sample in 0505_WaGa_wt 0505_WaGa_sT_DMSO 0505_WaGa_sT_Dox 0505_WaGa_scr_DMSO 0505_WaGa_scr_Dox 1905_WaGa_wt 1905_WaGa_sT_DMSO 1905_WaGa_sT_Dox 1905_WaGa_scr_DMSO 1905_WaGa_scr_Dox  control_MKL1 control_WaGa; do
     mkdir our_out/${sample}/
     java -jar COMPSRA.jar -ref hg38   -rh 20 -rt 20 -rr 20 -rlh 8,17 -aln -mt star -ann -ac 1,2,3,4,5,6  -in ${sample}2.fastq.gz -out ./our_out/
   done
   
   {miRNA}:
   [miRBase]
   Total Annotation Items: 4697
   Annotated Items (covered by least one read): 587
   Unannotated Items: 4110
   Reads Support the Annotation: 791636
   
   {piRNA}:
   [piRNABank]
   Total Annotation Items: 665175
   Annotated Items (covered by least one read): 480
   Unannotated Items: 664695
   Reads Support the Annotation: 6363051
   [piRBase]
   Total Annotation Items: 804849
   Annotated Items (covered by least one read): 1220
   Unannotated Items: 803629
   Reads Support the Annotation: 41374788
   
   {tRNA}:
   [GtRNAdb]
   Total Annotation Items: 601
   Annotated Items (covered by least one read): 440
   Unannotated Items: 161
   Reads Support the Annotation: 18690795
   
   {snoRNA}:
   [GEN_snoRNA]
   Total Annotation Items: 1006
   Annotated Items (covered by least one read): 250
   Unannotated Items: 756
   Reads Support the Annotation: 416228
   
   {snRNA}:
   [GEN_snRNA]
   Total Annotation Items: 2053
   Annotated Items (covered by least one read): 267
   Unannotated Items: 1786
   Reads Support the Annotation: 793559
   
   {circRNA}:
   [circRNA]
   Total Annotation Items: 140195
   Annotated Items (covered by least one read): 51488
   Unannotated Items: 88707
   Reads Support the Annotation: 14238651
   

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