Rick Masonbrink

Rick Masonbrink

Currently, I am an associate scientist in the Genome Informatics Facility at Iowa State University, working with a group of amazing people. Here, I work on a multitude of projects, including a continuation of my previous research. Some of these projects involve: characterizing the genomes of endangered abalone species, assessing the cellular roles of small RNAs in nematodes, creating user-friendly bioinformatics tutorials, assessing transposition in irradiated maize, trans-splicing in nematodes, etc. With the huge variation of collaborative projects that come into the Genome Informatics Facility, the constant influx of novel ideas creates an environment conducive to my development as a contributing member of the scientific community.

RSeQC tutorial to identify stranding information in RNAseq

RNA-seq technology has evolved and many datasets are now stranded, meaning strand specificity of origin for each transcript is retained in RNAseq.
Now comes the conundrum, what if this RNA-seq is not one you generated? There is always a need to confirm stranding information when not using self-generated data.

Install RSeQC

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#Attempt with miniconda did not succeeed.
ml miniconda2
conda create -n rseqc
source activate rseqc
conda install -c bioconda rseqc

# or do this
conda create -n rseqc
source activate rseqc
conda install -c bioconda bedops
pip install --user RSeQC

Generate bam files to assess for stranding information.

get the fastq files

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#get the fastq files
module load sra-toolkit

#unstranded samples
fastq-dump --outdir 01_Align/  --split-files SRR1573504 &
fastq-dump --outdir 01_Align/  --split-files SRR1573505 &

#stranded samples
fastq-dump --outdir 01_Align/  --split-files SRR13332812&
fastq-dump --outdir 01_Align/  --split-files SRR13332813 &

Get the genome and create the index

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#download the gene prediction .gff
wget https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/902/167/145/GCF_902167145.1_Zm-B73-REFERENCE-NAM-5.0/GCF_902167145.1_Zm-B73-REFERENCE-NAM-5.0_genomic.gff.gz
#download the genome and change the name to something simple.
wget https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/902/167/145/GCF_902167145.1_Zm-B73-REFERENCE-NAM-5.0/GCF_902167145.1_Zm-B73-REFERENCE-NAM-5.0_genomic.fna.gz
gunzip GCF_902167145.1_Zm-B73-REFERENCE-NAM-5.0_genomic.fna.gz
ln -s GCF_902167145.1_Zm-B73-REFERENCE-NAM-5.0_genomic.fna Maize.fa

#I am using hisat2 here, but you can use whatever aligner you like.
ml hisat2; hisat2-build Maize.fa Maize

Alignment script

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At this point we just assume that the reads are unstranded and align.  
If you have a huge number of reads that will take a significant amount of time to align, then take a subset of your fastq files
i.e. head -n 200000 R1.FASTQ >truncated_R1.fastq and head -n 200000 R2.fastq >truncated_R2.fastq

#runHiSat2bam.sh
################################################################################################################################
#note premake the database, hisat2-build genome.fa genome
#Script expects the database to be named the same as the genome file without the last extension (Genome), not (Genome.fa)
#Example run
#sh runHiSat2bam.sh {read1.fastq} {read2.fastq} {Directory of Database, not actual database without / at the end} {genomeName}

PROC=36
R1_FQ="$1"
R2_FQ="$2"
DBDIR="$3"
GENOME="$4"

module load hisat2
hisat2 -p ${PROC}  -x ${GENOME%.*} -1 ${R1_FQ} -2 ${R2_FQ}  -S ${R1_FQ%.*}.sam

module load samtools
samtools view --threads ${PROC} -b -o ${R1_FQ%.*}.bam ${R1_FQ%.*}.sam
mkdir ${R1_FQ%.*}_temp
samtools sort  -o ${R1_FQ%.*}_sorted.bam -T ${R1_FQ%.*}_temp --threads ${PROC} ${R1_FQ%.*}.bam
################################################################################################################################

Align

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This places the files in the order that the script requires: (sh runHiSat2bam.sh {read1.fastq} {read2.fastq} {Directory of Database, not actual database without / at the end} {genomeName})
paste <(ls -1 *_1.fastq) <(ls -1 *_2.fastq) |sed 's/\t/ /g' |while read line ; do echo "sh runHiSat2bam.sh "$line" /work/gif/TranscriptomicsWorkshop/remkv6/01_Maize/01_Align Maize.fa";done >Maizealign.sh
Maizealign.sh content 

```
sh runHiSat2bam.sh SRR13332812_1.fastq SRR13332812_2.fastq /work/gif/TranscriptomicsWorkshop/remkv6/01_Maize/01_Align Maize.fa
sh runHiSat2bam.sh SRR13332813_1.fastq SRR13332813_2.fastq /work/gif/TranscriptomicsWorkshop/remkv6/01_Maize/01_Align Maize.fa
sh runHiSat2bam.sh SRR1573504_1.fastq SRR1573504_2.fastq /work/gif/TranscriptomicsWorkshop/remkv6/01_Maize/01_Align Maize.fa
sh runHiSat2bam.sh SRR1573505_1.fastq SRR1573505_2.fastq /work/gif/TranscriptomicsWorkshop/remkv6/01_Maize/01_Align Maize.fa
```

Create bed12 file from genic gff

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#this converts your gff to bed12 format
ml bedops
gff2bed <GCF_902167145.1_Zm-B73-REFERENCE-NAM-5.0_genomic.gff >maizeGFF.bed12
maizeGFF.bed12 content 

```
NC_001666.2     0       140384  NC_001666.2:1..140384   .       +       RefSeq  region  .       ID=NC_001666.2:1..140384;Dbxref=taxon:4577;Is_circular=true;Name=Pltd;gbkey=Src;genome=chloroplast;mol_type=genomic DNA
NC_001666.2     0       140384  id-NC_001666.2:1..140384        .       +       RefSeq  sequence_feature        .       ID=id-NC_001666.2:1..140384;Note=[JLA]%3B junction IRA-LSC (circular molecule);gbkey=misc_feature
NC_001666.2     88      1150    cds-NP_043004.1 .       -       RefSeq  CDS     0       ID=cds-NP_043004.1;Parent=gene-ZemaCp002;Dbxref=UniProtKB/Swiss-Prot:P48183,Genbank:NP_043004.1,GeneID:845199;Name=NP_043004.1;gbkey=CDS;gene=psbA;locus_tag=ZemaCp002;product=photosystem II protein D1;protein_id=NP_043004.1;transl_table=11
NC_001666.2     88      1150    gene-ZemaCp002  .       -       RefSeq  gene    .       ID=gene-ZemaCp002;Dbxref=GeneID:845199;Name=psbA;gbkey=Gene;gene=psbA;gene_biotype=protein_coding;locus_tag=ZemaCp002
NC_001666.2     1385    1420    exon-ZemaCt121-2        .       -       RefSeq  exon    .       ID=exon-ZemaCt121-2;Parent=rna-ZemaCt121;Dbxref=GeneID:845256;gbkey=tRNA;gene=trnK;locus_tag=ZemaCt121;product=tRNA-Lys
NC_001666.2     1385    1420    id-ZemaCt121-2  .       -       RefSeq  exon    .       ID=id-ZemaCt121-2;Parent=gene-ZemaCt121;Dbxref=GeneID:845256;exon_number=2;gbkey=exon;gene=trnK;locus_tag=ZemaCt121;number=2
NC_001666.2     1385    3946    gene-ZemaCt121  .       -       RefSeq  gene    .       ID=gene-ZemaCt121;Dbxref=GeneID:845256;Name=trnK;gbkey=Gene;gene=trnK;gene_biotype=tRNA;locus_tag=ZemaCt121
NC_001666.2     1385    3946    rna-ZemaCt121   .       -       RefSeq  tRNA    .       ID=rna-ZemaCt121;Parent=gene-ZemaCt121;Dbxref=GeneID:845256;gbkey=tRNA;gene=trnK;locus_tag=ZemaCt121;product=tRNA-Lys
NC_001666.2     1420    3909    id-ZemaCt121    .       -       RefSeq  intron  .       ID=id-ZemaCt121;Parent=gene-ZemaCt121;Dbxref=GeneID:845256;exon_number=1;gbkey=intron;gene=trnK;locus_tag=ZemaCt121;number=1
NC_001666.2     1673    3215    cds-NP_043005.2 .       -       RefSeq  CDS     0       ID=cds-NP_043005.2;Parent=gene-ZemaCp003;Dbxref=UniProtKB/Swiss-Prot:P48190,Genbank:NP_043005.2,GeneID:845178;Name=NP_043005.2;gbkey=CDS;gene=matK;locus_tag=ZemaCp003;product=maturase K;protein_id=NP_043005.2;transl_table=11
NC_001666.2     1673    3215    gene-ZemaCp003  .       -       RefSeq  gene    .       ID=gene-ZemaCp003;Dbxref=GeneID:845178;Name=matK;gbkey=Gene;gene=matK;gene_biotype=protein_coding;locus_tag=ZemaCp003
NC_001666.2     3909    3946    exon-ZemaCt121-1        .       -       RefSeq  exon    .       ID=exon-ZemaCt121-1;Parent=rna-ZemaCt121;Dbxref=GeneID:845256;gbkey=tRNA;gene=trnK;locus_tag=ZemaCt121;product=tRNA-Lys
NC_001666.2     3909    3946    id-ZemaCt121-1  .       -       RefSeq  exon    .       ID=id-ZemaCt121-1;Parent=gene-ZemaCt121;Dbxref=GeneID:845256;exon_number=1;gbkey=exon;gene=trnK;locus_tag=ZemaCt121;number=1
NC_001666.2     4490    4708    cds-NP_043006.1 .       -       RefSeq  CDS     2       ID=cds-NP_043006.1;Parent=gene-ZemaCp004;Dbxref=UniProtKB/Swiss-Prot:P27723,Genbank:NP_043006.1,GeneID:845232;Name=NP_043006.1;gbkey=CDS;gene=rps16;locus_tag=ZemaCp004;product=ribosomal protein S16;protein_id=NP_043006.1;transl_table=11
NC_001666.2     4490    4708    id-ZemaCp004-2  .       -       RefSeq  exon    .       ID=id-ZemaCp004-2;Parent=gene-ZemaCp004;Dbxref=GeneID:845232;exon_number=2;gbkey=exon;gene=rps16;locus_tag=ZemaCp004;number=2
NC_001666.2     4490    5604    gene-ZemaCp004  .       -       RefSeq  gene    .       ID=gene-ZemaCp004;Dbxref=GeneID:845232;Name=rps16;gbkey=Gene;gene=rps16;gene_biotype=protein_coding;locus_tag=ZemaCp004
NC_001666.2     4708    5564    id-ZemaCp004    .       -       RefSeq  intron  .       ID=id-ZemaCp004;Parent=gene-ZemaCp004;Dbxref=GeneID:845232;exon_number=1;gbkey=intron;gene=rps16;locus_tag=ZemaCp004;number=1
NC_001666.2     5564    5604    cds-NP_043006.1 .       -       RefSeq  CDS     0       ID=cds-NP_043006.1;Parent=gene-ZemaCp004;Dbxref=UniProtKB/Swiss-Prot:P27723,Genbank:NP_043006.1,GeneID:845232;Name=NP_043006.1;gbkey=CDS;gene=rps16;locus_tag=ZemaCp004;product=ribosomal protein S16;protein_id=NP_043006.1;transl_table=11
NC_001666.2     5564    5604    id-ZemaCp004-1  .       -       RefSeq  exon    .       ID=id-ZemaCp004-1;Parent=gene-ZemaCp004;Dbxref=GeneID:845232;exon_number=1;gbkey=exon;gene=rps16;locus_tag=ZemaCp004;number=1
NC_001666.2     6772    6844    exon-ZemaCt122-1        .       -       RefSeq  exon    .       ID=exon-ZemaCt122-1;Parent=rna-ZemaCt122;Dbxref=GeneID:845265;gbkey=tRNA;gene=trnQ;locus_tag=ZemaCt122;product=tRNA-Gln
NC_001666.2     6772    6844    gene-ZemaCt122  .       -       RefSeq  gene    .       ID=gene-ZemaCt122;Dbxref=GeneID:845265;Name=trnQ;gbkey=Gene;gene=trnQ;gene_biotype=tRNA;locus_tag=ZemaCt122
NC_001666.2     6772    6844    rna-ZemaCt122   .       -       RefSeq  tRNA    .       ID=rna-ZemaCt122;Parent=gene-ZemaCt122;Dbxref=GeneID:845265;gbkey=tRNA;gene=trnQ;locus_tag=ZemaCt122;product=tRNA-Gln
NC_001666.2     7198    7384    cds-NP_043007.1 .       +       RefSeq  CDS     0       ID=cds-NP_043007.1;Parent=gene-ZemaCp005;Dbxref=UniProtKB/Swiss-Prot:P48188,Genbank:NP_043007.1,GeneID:845208;Name=NP_043007.1;gbkey=CDS;gene=psbK;locus_tag=ZemaCp005;product=photosystem II protein K;protein_id=NP_043007.1;transl_table=11
NC_001666.2     7198    7384    gene-ZemaCp005  .       +       RefSeq  gene    .       ID=gene-ZemaCp005;Dbxref=GeneID:845208;Name=psbK;gbkey=Gene;gene=psbK;gene_biotype=protein_coding;locus_tag=ZemaCp005
NC_001666.2     7774    7885    cds-NP_043008.1 .       +       RefSeq  CDS     0       ID=cds-NP_043008.1;Parent=gene-ZemaCp006;Dbxref=UniProtKB/Swiss-Prot:P09970,Genbank:NP_043008.1,GeneID:845206;Name=NP_043008.1;gbkey=CDS;gene=psbI;locus_tag=ZemaCp006;product=photosystem II protein I;protein_id=NP_043008.1;transl_table=11
NC_001666.2     7774    7885    gene-ZemaCp006  .       +       RefSeq  gene    .       ID=gene-ZemaCp006;Dbxref=GeneID:845206;Name=psbI;gbkey=Gene;gene=psbI;gene_biotype=protein_coding;locus_tag=ZemaCp006
NC_001666.2     8011    8099    exon-ZemaCt123-1        .       -       RefSeq  exon    .       ID=exon-ZemaCt123-1;Parent=rna-ZemaCt123;Dbxref=GeneID:845270;gbkey=tRNA;gene=trnS;locus_tag=ZemaCt123;product=tRNA-Ser
NC_001666.2     8011    8099    gene-ZemaCt123  .       -       RefSeq  gene    .       ID=gene-ZemaCt123;Dbxref=GeneID:845270;Name=trnS;gbkey=Gene;gene=trnS;gene_biotype=tRNA;locus_tag=ZemaCt123
NC_001666.2     8011    8099    rna-ZemaCt123   .       -       RefSeq  tRNA    .       ID=rna-ZemaCt123;Parent=gene-ZemaCt123;Dbxref=GeneID:845270;gbkey=tRNA;gene=trnS;locus_tag=ZemaCt123;product=tRNA-Ser
NC_001666.2     9082    10144   cds-NP_043009.1 .       +       RefSeq  CDS     0       ID=cds-NP_043009.1;Parent=gene-ZemaCp007;Dbxref=UniProtKB/Swiss-Prot:P48184,Genbank:NP_043009.1,GeneID:845202;Name=NP_043009.1;gbkey=CDS;gene=psbD;locus_tag=ZemaCp007;product=photosystem II protein D2;protein_id=NP_043009.1;transl_table=11
NC_001666.2     9082    10144   gene-ZemaCp007  .       +       RefSeq  gene    .       ID=gene-ZemaCp007;Dbxref=GeneID:845202;Name=psbD;gbkey=Gene;gene=psbD;gene_biotype=protein_coding;locus_tag=ZemaCp007
NC_001666.2     10091   11513   cds-NP_043010.1 .       +       RefSeq  CDS     0       ID=cds-NP_043010.1;Parent=gene-ZemaCp008;Dbxref=UniProtKB/Swiss-Prot:P48187,Genbank:NP_043010.1,GeneID:845201;Name=NP_043010.1;Note=CP43;gbkey=CDS;gene=psbC;locus_tag=ZemaCp008;product=photosystem II 44 kDa protein;protein_id=NP_043010.1;transl_table=11
NC_001666.2     10091   11513   gene-ZemaCp008  .       +       RefSeq  gene    .       ID=gene-ZemaCp008;Dbxref=GeneID:845201;Name=psbC;gbkey=Gene;gene=psbC;gene_biotype=protein_coding;locus_tag=ZemaCp008
NC_001666.2     11668   11756   exon-ZemaCt124-1        .       -       RefSeq  exon    .       ID=exon-ZemaCt124-1;Parent=rna-ZemaCt124;Dbxref=GeneID:845271;gbkey=tRNA;gene=trnS;locus_tag=ZemaCt124;product=tRNA-Ser
NC_001666.2     11668   11756   gene-ZemaCt124  .       -       RefSeq  gene    .       ID=gene-ZemaCt124;Dbxref=GeneID:845271;Name=trnS;gbkey=Gene;gene=trnS;gene_biotype=tRNA;locus_tag=ZemaCt124
NC_001666.2     11668   11756   rna-ZemaCt124   .       -       RefSeq  tRNA    .       ID=rna-ZemaCt124;Parent=gene-ZemaCt124;Dbxref=GeneID:845271;gbkey=tRNA;gene=trnS;locus_tag=ZemaCt124;product=tRNA-Ser
NC_001666.2     12016   12205   cds-NP_043011.1 .       +       RefSeq  CDS     0       ID=cds-NP_043011.1;Parent=gene-ZemaCp009;Dbxref=UniProtKB/Swiss-Prot:Q33300,Genbank:NP_043011.1,GeneID:1466358;Name=NP_043011.1;Note=YCF9;gbkey=CDS;gene=psbZ;locus_tag=ZemaCp009;product=photosystem II protein Z;protein_id=NP_043011.1;transl_table=11
NC_001666.2     12016   12205   gene-ZemaCp009  .       +       RefSeq  gene    .       ID=gene-ZemaCp009;Dbxref=GeneID:1466358;Name=psbZ;gbkey=Gene;gene=psbZ;gene_biotype=protein_coding;locus_tag=ZemaCp009
NC_001666.2     12508   12579   exon-ZemaCt125-1        .       +       RefSeq  exon    .       ID=exon-ZemaCt125-1;Parent=rna-ZemaCt125;Dbxref=GeneID:845248;gbkey=tRNA;gene=trnG;locus_tag=ZemaCt125;product=tRNA-Gly
NC_001666.2     12508   12579   gene-ZemaCt125  .       +       RefSeq  gene    .       ID=gene-ZemaCt125;Dbxref=GeneID:845248;Name=trnG;gbkey=Gene;gene=trnG;gene_biotype=tRNA;locus_tag=ZemaCt125
NC_001666.2     12508   12579   rna-ZemaCt125   .       +       RefSeq  tRNA    .       ID=rna-ZemaCt125;Parent=gene-ZemaCt125;Dbxref=GeneID:845248;gbkey=tRNA;gene=trnG;locus_tag=ZemaCt125;product=tRNA-Gly
NC_001666.2     12985   12986   id-NC_001666.2:12986..12986     .       +       RefSeq  sequence_conflict       .       ID=id-NC_001666.2:12986..12986;gbkey=conflict;replace=;zero_length_insertion=True
NC_001666.2     12998   12999   id-NC_001666.2:12999..12999     .       +       RefSeq  sequence_conflict       .       ID=id-NC_001666.2:12999..12999;gbkey=conflict;replace=a;zero_length_insertion=True
NC_001666.2     13072   13146   exon-ZemaCt126-1        .       -       RefSeq  exon    .       ID=exon-ZemaCt126-1;Parent=rna-ZemaCt126;Dbxref=GeneID:845280;gbkey=tRNA;gene=trnfM;locus_tag=ZemaCt126;product=tRNA-Met
NC_001666.2     13072   13146   gene-ZemaCt126  .       -       RefSeq  gene    .       ID=gene-ZemaCt126;Dbxref=GeneID:845280;Name=trnfM;gbkey=Gene;gene=trnfM;gene_biotype=tRNA;locus_tag=ZemaCt126
NC_001666.2     13072   13146   rna-ZemaCt126   .       -       RefSeq  tRNA    .       ID=rna-ZemaCt126;Parent=gene-ZemaCt126;Dbxref=GeneID:845280;gbkey=tRNA;gene=trnfM;locus_tag=ZemaCt126;product=tRNA-Met
NC_001666.2     13244   13292   exon-ZemaCt127-2        .       -       RefSeq  exon    .       ID=exon-ZemaCt127-2;Parent=rna-ZemaCt127;Dbxref=GeneID:845249;gbkey=tRNA;gene=trnG;locus_tag=ZemaCt127;product=tRNA-Gly
NC_001666.2     13244   13292   id-ZemaCt127-2  .       -       RefSeq  exon    .       ID=id-ZemaCt127-2;Parent=gene-ZemaCt127;Dbxref=GeneID:845249;exon_number=2;gbkey=exon;gene=trnG;locus_tag=ZemaCt127;number=2
NC_001666.2     13244   14013   gene-ZemaCt127  .       -       RefSeq  gene    .       ID=gene-ZemaCt127;Dbxref=GeneID:845249;Name=trnG;gbkey=Gene;gene=trnG;gene_biotype=tRNA;locus_tag=ZemaCt127
NC_001666.2     13244   14013   rna-ZemaCt127   .       -       RefSeq  tRNA    .       ID=rna-ZemaCt127;Parent=gene-ZemaCt127;Dbxref=GeneID:845249;gbkey=tRNA;gene=trnG;locus_tag=ZemaCt127;product=tRNA-Gly
NC_001666.2     13292   13990   id-ZemaCt127    .       -       RefSeq  intron  .       ID=id-ZemaCt127;Parent=gene-ZemaCt127;Dbxref=GeneID:845249;exon_number=1;gbkey=intron;gene=trnG;locus_tag=ZemaCt127;number=1
NC_001666.2     13990   14013   exon-ZemaCt127-1        .       -       RefSeq  exon    .       ID=exon-ZemaCt127-1;Parent=rna-ZemaCt127;Dbxref=GeneID:845249;gbkey=tRNA;gene=trnG;locus_tag=ZemaCt127;product=tRNA-Gly
NC_001666.2     13990   14013   id-ZemaCt127-1  .       -       RefSeq  exon    .       ID=id-ZemaCt127-1;Parent=gene-ZemaCt127;Dbxref=GeneID:845249;exon_number=1;gbkey=exon;gene=trnG;locus_tag=ZemaCt127;number=1
NC_001666.2     14497   14707   cds-NP_043012.1 .       -       RefSeq  CDS     0       ID=cds-NP_043012.1;Parent=gene-ZemaCp010;Dbxref=UniProtKB/TrEMBL:Q33301,Genbank:NP_043012.1,GeneID:1466359;Name=NP_043012.1;Note=ORF69;gbkey=CDS;locus_tag=ZemaCp010;product=hyp
etc...
```

run RSeQC

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#assesses the strand information of your alignment
for f in *sorted.bam; do infer_experiment.py --i $f -r maizeGFF.bed12 ;done
Printed content of the above for loop 

```
infer_experiment.py --i SRR13332812_1_sorted.bam -r maizeGFF.bed12
infer_experiment.py --i SRR13332813_1_sorted.bam -r maizeGFF.bed12
infer_experiment.py --i SRR1573504_1Gene_sorted.bam -r maizeGFF.bed12
infer_experiment.py --i SRR1573505_1Gene_sorted.bam -r maizeGFF.bed12
```

Results for unstranded reads

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[E::idx_find_and_load] Could not retrieve index file for 'SRR1573504_1Gene_sorted.bam'
Reading reference gene model maizeGFF.bed12 ... Done
Loading SAM/BAM file ...  Total 200000 usable reads were sampled


This is PairEnd Data
Fraction of reads failed to determine: 0.5082
Fraction of reads explained by "1++,1--,2+-,2-+": 0.2406
Fraction of reads explained by "1+-,1-+,2++,2--": 0.2512
[E::idx_find_and_load] Could not retrieve index file for 'SRR1573505_1Gene_sorted.bam'
Reading reference gene model maizeGFF.bed12 ... Done
Loading SAM/BAM file ...  Total 200000 usable reads were sampled


This is PairEnd Data
Fraction of reads failed to determine: 0.3724
Fraction of reads explained by "1++,1--,2+-,2-+": 0.3053
Fraction of reads explained by "1+-,1-+,2++,2--": 0.3222

Results for stranded reads

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[E::idx_find_and_load] Could not retrieve index file for 'SRR13332812_1_sorted.bam'
Reading reference gene model maizeGFF.bed12 ... Done
Loading SAM/BAM file ...  Total 200000 usable reads were sampled


This is PairEnd Data
Fraction of reads failed to determine: 0.4494
Fraction of reads explained by "1++,1--,2+-,2-+": 0.0349
Fraction of reads explained by "1+-,1-+,2++,2--": 0.5157
[E::idx_find_and_load] Could not retrieve index file for 'SRR13332813_1_sorted.bam'
Reading reference gene model maizeGFF.bed12 ... Done
Loading SAM/BAM file ...  Total 200000 usable reads were sampled


This is PairEnd Data
Fraction of reads failed to determine: 0.4496
Fraction of reads explained by "1++,1--,2+-,2-+": 0.0331
Fraction of reads explained by "1+-,1-+,2++,2--": 0.5174

We see that the stranded reads have an RF orientation and can make adjustments in the aligner to align these reads using strand information.

References

RSeQC Sourceforge