Dts-seq

Computational workflow for Direct tRNA sequencing (Dts-seq) analysis

Citation: Accurate characterization of Escherichia coli tRNA modifications with a simple method of deep sequencing library preparation. Wang J, Toffano-Nioche C, Lorieux F, Gautheret D, Lehmann J. RNA Biol. 2020 Jul 3. doi: 10.1080/15476286.2020.1790871. PMID: 32618488

Contacts

• Claire Toffano-Nioche (claire.toffano-nioche@u-psud.fr)
• Daniel Gautheret (daniel.gautheret@u-psud.fr)
• Jean Lehmann (jean.lehmann@u-psud.fr)

Note about installing the third-party tools

In order to increase the reproducibility of the computational analyses, we used the docker solution as much as possible. If you haven't yet docker, you may follow the installation page (docker) or install the third-party tools listed at the end of this document.

RNAseq: from fastq to read coverage

Repository architecture

• Protocol: creating a repository for the analysis:
• Code:

# download and decompress, or clone codes & data
git clone https://github.com/i2bc/dts-seq.git
# create the Dts-seq repositories & links with the codes
cd dts-seq ; 
DTSSEQDIR=$(pwd) ;
mkdir 1_rawData 2_processedData 3_mapping 4_selection 5_coverage 6_tRNA_modification 7_termination_signal
mkdir 2_processedData/FastQC 3_mapping/index_bt2x
mv NC_000913_tRNA.tsv 6_tRNA_modification/.
mv bmModomics_with_tmRNA.fasta 6_tRNA_modification/.
mv bmModomics.txt 6_tRNA_modification/.
mv trnaprint.txt 7_termination_signal/.

• Result: the architecture of dts-seq repository

.
├── 1_rawData
├── 2_processedData
│   └── FastQC
├── 3_mapping
│   └── index_bt2x
├── 4_selection
├── 5_coverage
├── 6_tRNA_modification
│   ├── bmModomics.txt
│   ├── bmModomics_with_tmRNA.fasta
│   └── NC_000913_tRNA.tsv
├── 7_termination_signal
│   └── trnaprint.txt
├── dts-seq-plot.r
├── GCF_000005845.2_ASM584v2_genomic.fna
├── README.md
└── tRNA_features.txt

Data

Genome & annotations

• Protocol: download genome from ncbi, accession: GCF_000005845.2_ASM584v2
• Code :

cd 1_rawData
wget ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/005/845/GCF_000005845.2_ASM584v2/GCF_000005845.2_ASM584v2_genomic.fna.gz
wget ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/005/845/GCF_000005845.2_ASM584v2/GCF_000005845.2_ASM584v2_genomic.gff.gz
gunzip GCF_000005845.2_ASM584v2_genomic.*.gz
cd ..

• Result files (into dts-seq/1_rawData repository):
  • GCF_000005845.2_ASM584v2_genomic.fna
  • GCF_000005845.2_ASM584v2_genomic.gff

RNAseq Data

• Protocol: R2 files were downloaded from ENA (access: PRJEB33277) into the local dts-seq/1_rawData repository.
• Code to list all samples files:

cd 1_rawData ;
for rep in A B C ; do 
    for samples in 3-D 4-NT 5-nD ; do 
        wget ${rep}${samples}_R2.fastq.gz ; 
    done ; 
done ;
cd ..

• Result files: 9 *_R2_fastq.gz

read number	A5-nD, B5-nD, C5-nD	A4-NT, B4-NT, C4-NT	A3-D, B3-D, C3-D
QC-passed	11796813, 13114509, 11455875	11680073, 13517533, 10775965	15388902, 12562074, 10691188

Cleaning

• Protocol: each raw fastq file was cleaned (cutadapt) with a specific polyA adapter following the wet protocol and reads shorter than 10 bp after polyA trimming were discarded. Quality control was performed (FastQC software).
• Code:

rm 2_processedData/cutadapt.log ; rm 2_processedData/fastqc.log ;
for i in `ls 1_rawData/*_R2.fastq.gz` ; do 
   SampleName=`basename -s _R2.fastq.gz ${i}` ; 
   docker run -v ${DTSSEQDIR}:/data:rw -w /data docker-registry.genouest.org/bioconda/cutadapt cutadapt --adapter=AAAAAAAAAAAAAAA --minimum-length=10 --output=2_processedData/${SampleName}_noPolyA.fastq ${i} >> 2_processedData/cutadapt.log 2>&1 ;
   docker run -v ${DTSSEQDIR}:/data:rw -w /data docker-registry.genouest.org/ifb/fastqc fastqc -o 2_processedData/FastQC 2_processedData/${SampleName}_noPolyA.fastq >> 2_processedData/FastQC/fastqc.log 2>&1 ;
done

• Result files (into 2_processedData repository):
  • 9 output fastq files (*_noPolyA.fastq)
  • 2 log files (cutadapt.log, fastqc.log)
  • 9 repositories containing the quality control results (*.html)

read number	A5-nD, B5-nD, C5-nD	A4-NT, B4-NT, C4-NT	A3-D, B3-D, C3-D
trimmed	9736417, 8639091, 9522279	10257977, 10586494, 10181006	12595828, 10640726, 9146981

Mapping step

• Protocol: reads mapping was done by bowtie2 with the local mapping option up to maximize the alignment length.
• Code:

# create genome index file for bowtie2
rm 3_mapping/bowtie2-build.log ; rm 3_mapping/bowtie2-align.log ;
docker run -v ${DTSSEQDIR}:/data:rw -w /data docker-registry.genouest.org/bioconda/bowtie2 bowtie2-build 1_rawData/GCF_000005845.2_ASM584v2_genomic.fna 3_mapping/index_bt2x/NC_00913 >> 3_mapping/bowtie2-build.log 2>&1
# bowtie2 run
for i in 2_processedData/*_noPolyA.fastq ; do 
   sample=`basename $i _noPolyA.fastq` ; 
   docker run -v ${DTSSEQDIR}:/data:rw -w /data docker-registry.genouest.org/bioconda/bowtie2 bowtie2 -x 3_mapping/index_bt2x/NC_00913 --phred33 --local $i > 3_mapping/${sample}.sam 2>> 3_mapping/bowtie2-align.log ; 
done

• Result files (into 3_mapping repository):
  • 6 index files for bowtie2 (*.btz2 into index_bt2x repository)
  • 9 *.sam
  • log files: bowtie2-align.log, bowtie2-build.log

read number	A5-nD, B5-nD, C5-nD	A4-NT, B4-NT, C4-NT	A3-D, B3-D, C3-D
mapped	7102322, 5656027, 7442976	7767199, 7449606, 8113918	9712034, 8033555, 7173882

Alignment selection

• Protocol: selection of mapped reads on the genomic sequence and presenting a complete 3' end, ie. either CCA or TGG depending on the DNA strand (awk). The resulting alignment files were sorted by increasing locations and the associated index files for binary management were created.
• Code:

rm 4_selection/selection.log
for rep in A B C ; do for s in "3-D" "4-N"T "5-nD" ; do
   awk 'BEGIN{FS="\t";OFS="\t"}{if( ($0~/@/)||((($2==16)&&($10~/CCA$/))||(($2==0)&&($10~/^TGG/))) ){print $0}}' 3_mapping/${rep}${s}.sam | samtools view -hu - | samtools sort - > 4_selection/${rep}${s}_CCATGG.bam 2>> 4_selection/selection.log ; 
   samtools index 4_selection/${rep}${s}_CCATGG.bam 2>> 4_selection/selection.log ;
done ; done ;

• Result files (into 4_selection repository):
  • 9 *_CCATGG.bam
  • 9 *_CCATGG.bam.bai

read number	A5-nD, B5-nD, C5-nD	A4-NT, B4-NT, C4-NT	A3-D, B3-D, C3-D
remaining	6505322, 5161234, 6950080	7236088, 6816582, 7567646	9051878, 7466730, 6781275

Read coverage computation

• Protocol: creation of coverage files (both format wig and 2 columns) with strand separation. As alignments came from R2 reads, exchange of reverse and forward strands (join).
• Code:

for i in 4_selection/*_CCATGG.bam ; do 
   sample=`basename $i .bam` ; 
   # coverage of reverse strand
   samtools view -h -b -f 16 ${i} NC_000913.3 | samtools depth -d 10000000 -a - > 5_coverage/${sample}_depth_rev.txt ; 
   # coverage for forward strand
   samtools view -h -b -F 0x14 ${i} NC_000913.3 | samtools depth -d 10000000 -a - > 5_coverage/${sample}_depth_for.txt ; 
   # strands association 
   join -t $'\t' -12 -22 -o 1.3,2.3 5_coverage/${sample}_depth_rev.txt 5_coverage/${sample}_depth_for.txt > 5_coverage/${sample}_depth_fr.txt ;
done 

• Result files (into 5_coverage repository):
  • 9 *_depth_for.txt (coverage on forward strand)
  • 9 *_depth_rev.txt (coverage on reverse strand)
  • 9 *_depth_fr.txt (coverage on both strands)

tRNA modified bases from Modomics DB

Data

• Protocol: sequences with modified bases were get from modomics DB for the Escherichia coli specie, acces: clic on "Display as ASCII" buton and copy/paste in text format file (downloaded on november 2017, bmModomics.txt). Manually apply 2 modifications: i) deduplicate 4 tRNA names for Ini_CAU, Thr_GGU, Tyr_QUA, Val_GAC, and ii) duplicate the "_" character of selC following the footnote of the Modomics page. Create 3 fasta files from bmModomics.txt: i) without modified bases (bmModomics_seqU.fasta), ii) without any bases but modified ones (bmModomics_noBM.fasta), and iii) 43 tRNA DNA sequences without any modified base (tRNA_coli.fasta).
• Code:

sed 's/-//g;s/> tRNA/>tRNA/g;s/ | Escherichia coli | prokaryotic cytosol//g;s/ | /_/g;' 6_tRNA_modification/bmModomics.txt > 6_tRNA_modification/bmModomics_seqU.fasta
sed 'n;s/[AGCU_]/ /g' 6_tRNA_modification/bmModomics_seqU.fasta > 6_tRNA_modification/bmModomics_noBM.fasta
sed 'n;y!=/){}$*#%+⊄467BDEIJKMPQSTVX!AAUUCUAGCANUAGCUANUGCUNUUUU!;' 6_tRNA_modification/bmModomics_seqU.fasta | sed 's/-//g;s/U/T/g;s/⊄/0/g' > 6_tRNA_modification/tRNA_coli.fasta

• Result files (in 6_tRNA_modification repository):
  • downloaded file with 43 tRNA sequences including knowed modified bases, bmModomics.txt
  • without any bases but modified ones (bmModomics_noBM.fasta)
  • without modified bases (bmModomics_seqU.fasta)
  • DNA sequences without modified bases (tRNA_coli.fasta)

Genomic coordinates of modified bases

• Protocol: alignment (blastn software) of the modomics tRNA sequences to the genomic sequence. Manual analysis of the blastn report in order to associate the genomic locations to the 43 tRNA of modomics and to create the tRNA groups for those that have the same genomic sequence. The resulting file contains 91 tRNA regions (including pseudo-tRNAs and the ssrA-tmRNA).
• Code:

rm 6_tRNA_modification/blastDB-build.log ; rm 6_tRNA_modification/blastn-run.log
# create blastDB:
docker run -v ${DTSSEQDIR}:/in:rw -w /in chrishah/ncbi-blast:v2.6.0 makeblastdb -in 6_tRNA_modification/tRNA_coli.fasta -out 6_tRNA_modification/tRNA_BD -parse_seqids -dbtype nucl >> 6_tRNA_modification/blastDB-build.log 2>&1
# run blastn:
docker run -v ${DTSSEQDIR}:/in:rw -w /in chrishah/ncbi-blast:v2.6.0 blastn -out 6_tRNA_modification/blastn_table -query 1_rawData/GCF_000005845.2_ASM584v2_genomic.fna -db 6_tRNA_modification/tRNA_DB -outfmt 7 >> 6_tRNA_modification/blastn-run.log 2>&1

• Result files (in 6_tRNA_modification repository):
  • the blastDB files (6 tRNA_DB.n* files)
  • the blastn report (blastn_table)
  • tRNA_features.txt (manually designed from the blastn report)

Termination signal: from read coverage to ts-jump

• Protocol: with information from the tRNA_features.txt file and the read coverage computation for each sample, compute and plot the "ts-signal".
• Code (launch R into the dts-seq repository):

source("dts-seq-plot.r")

Softwares version used

• docker version 18.09.2, build 6247962
• fastqc: from docker-registry.genouest.org/ifb/fastqc, version 0.11.5
• cutadapt: from docker-registry.genouest.org/bioconda/cutadapt, version 1.11
• bowtie2: from docker-registry.genouest.org/bioconda/bowtie2, bowtie2-align-s version 2.2.8
• samtools: version 1.4
• blast: from the ncbi-blast-2.6.0+ version
• R: version 3.4.4