Orient reads as per protocol Preparing Reads for Stranded Mapping.

If this has been done, then the following command should produce output without errors:

for bc in $(awk '{print $2}' barcode_counts.txt);
  do ls oriented/${bc}_reads_dirAdjusted.fq.gz;
done
```Example output:

oriented/BC03_reads_dirAdjusted.fq.gz oriented/BC04_reads_dirAdjusted.fq.gz oriented/BC05_reads_dirAdjusted.fq.gz oriented/BC06_reads_dirAdjusted.fq.gz oriented/BC07_reads_dirAdjusted.fq.gz oriented/BC08_reads_dirAdjusted.fq.gz

Prepare genome index for spliced alignment

Software

Value	Label
minimap2	NAME
Linux	OS_NAME
http://github.com/lh3/minimap2	REPOSITORY
Heng Li	DEVELOPER
http://github.com/lh3/minimap2/releases	LINK
2.16	VERSION

minimap2 -d mmus_ucsc_all-splice.idx -Q -t 10 -x splice mmus_ucsc_all.fa

Map the long reads to the genome using minimap2, using samtools to covert to a sorted BAM format. This is where the reverse complementing done during demultiplexing gives a big saving of effort. As this BAM file is one of the main outputs, the run name is added to the file name.

Software

Value	Label
SAMtools	NAME
Linux	OS_NAME
http://github.com/samtools/samtools	REPOSITORY
Wellcome Trust Sanger Institute	DEVELOPER
http://www.htslib.org/download/	LINK
1.8	VERSION

runName="CHANGE_THIS";
mkdir -p mapped;
for bc in $(awk '{print $2}' barcode_counts.txt);
  do echo ${bc};
  minimap2 -t 10 -a -x splice mmus_ucsc_all-splice.idx oriented/${bc}_reads_dirAdjusted.fq.gz | \
    samtools view -b | samtools sort > mapped/mm2_${runName}_called_${bc}_vs_MmusG.bam;
done

mpileupDC.pl

A bedGraph of coverage is created using samtools mpileup and mpileupDC.pl, excluding any skipped intronic sequence. When 'mpileupDC.pl' is provided with a single file, it will output a bedGraph file with a header line starting with '##'; this header line is removed.

To simplify output naming in later steps, the run name is added to the file name.

The particular JBrowse plugin that I use for stranded display requires that the reverse strand have negative coverage values, so that file needs to be changed:

runName="CHANGE_THIS";
for bc in $(awk '{print $2}' barcode_counts.txt);
  do echo ${bc};
  samtools view -b -F 0x10 mapped/mm2_called_${bc}_vs_MmusG.bam | \
    samtools mpileup -A -B -Q 0 -q 0 -I -q 0 -Q 0 - | \
    mpileupDC.pl | tail -n +2 > mapped/mm2_${runName}_called_${bc}_vs_MmusG.bg.plus
  samtools view -b -f 0x10 mapped/mm2_called_${bc}_vs_MmusG.bam | \
    samtools mpileup -A -B -Q 0 -q 0 -I -q 0 -Q 0 - | \
    mpileupDC.pl | tail -n +2 > mapped/mm2_${runName}_called_${bc}_vs_MmusG.bg.minus
  perl -i -pe 's/([0-9]+)$/-$1/' mapped/mm2_${runName}_called_${bc}_vs_MmusG.bg.minus
done;
```As an alternative, BEDTools can be used to generate coverage. The default options for BEDTools treat sequence deletions (which happen frequently in nanopore reads) as a drop in coverage, which can make exon hunting and coverage calculation more difficult. I submitted [a pull request](http://github.com/arq5x/bedtools2/pull/868) to add an additional  _ignoreD_  parameter to the command line to allow cDNA reads with split coverage across introns to ignore deletions when considering coverage; this request has now been incorporated into the main BEDtools repository (as of v2.30.0):




Software

| Value | Label |
| --- | --- |
| BEDtools | NAME |
| Linux | OS_NAME |
| http://github.com/arq5x/bedtools2 | REPOSITORY |
| Aaron Quinlan | DEVELOPER |
| http://github.com/arq5x/bedtools2/releases/tag/v2.30.0 | LINK |
| 2.30.0 | VERSION |

runName="CHANGE_THIS"; for bc in 2}' barcode_counts.txt); do echo {runName}called{runName}called{runName}called{runName}called/-{runName}called${bc}_vs_MmusG.bg.minus done;

Stranded bedgraph files are converted to bigwig. This requires BEDTools and a genome information file containing chromosome lengths (one for Mmus/mm10 is attached to this step). As this bigwig file is one of the main outputs, the run name is added to the file name.

runName="CHANGE_THIS";
for bc in $(awk '{print $2}' barcode_counts.txt);
  do echo ${bc};
  basename="mapped/mm2_${runName}_called_${bc}_vs_MmusG"
  bedGraphToBigWig ${basename}.bg.plus Mmus_genome.chrInfo.txt ${basename}.bw.plus
  bedGraphToBigWig ${basename}.bg.minus Mmus_genome.chrInfo.txt ${basename}.bw.minus 
done
```[Mmus_genome.chrInfo.txt](http://static.aivcloud.com/literature_test/protocol_io_true/protocols.io.b54iq8ue/bgevjpt61.txt)

Each track should have its own JBrowse configuration section using the StrangedBigWig class and StrandedXYPlot type. BAM tracks can also be added. An example is shown here:

[tracks.bam-CG005_Nov18_BC03-track]
# settings for what data is shown in the track
storeClass     = JBrowse/Store/SeqFeature/BAM
urlTemplate    = raw/mm2_called_CG005AB_BC03_vs_MmusG.bam
baiUrlTemplate = raw/mm2_called_CG005AB_BC03_vs_MmusG.bam.bai
chunkSizeLimit = 10000000
maxHeight      = 3000
# settings for how the track looks
category = MinION / Alignments
type = JBrowse/View/Track/Alignments2
key  = Minimap2 alignments from 4T1.ρ0#C [CG005]

[tracks.bw-CG005_Nov18_BC03-both-track]
storeClass     = StrandedPlotPlugin/Store/SeqFeature/StrandedBigWig
urlTemplate    = bw/mm2_called_CG005AB_BC03_vs_MmusG.bw
category       = MinION / Coverage
type           = StrandedPlotPlugin/View/Track/Wiggle/StrandedXYPlot
key            = Minimap2 coverage from 4T1.ρ0#C [CG005]
scale          = log
scoreType      = maxScore
autoscale      = global
style.pos_color = #228B22
style.neg_color = lightskyblue

[tracks.BWCG004-4T1-BC04-both-track ] storeClass = StrandedPlotPlugin/Store/SeqFeature/StrandedBigWig urlTemplate = bw/mm2_called_CG004_BC04_vs_MmusG.bw category = MinION - Coverage type = StrandedPlotPlugin/View/Track/Wiggle/StrandedXYPlot key = MinION minimap2 coverage from CG004-4T1-WT (combined strands) scale = log scoreType = maxScore autoscale = global style.pos_color = darkred style.neg_color = darkgreen

./makeMinIONTemplate.sh mm2_called_CG005AB_BC03_vs_MmusG CG005_Nov18_BC03 '4T1.ρ0#C [CG005]' >> tracks.conf

[makeMinIONTemplate.sh](http://static.aivcloud.com/literature_test/protocol_io_true/protocols.io.b54iq8ue/cnn7jpt63.sh) 

If this has worked properly, then mapping human or mouse to the mitochondrial genome should show most expression appearing on the positive strand, with a small scattering of negative-strand expression, a bit like the Expected Results shown here.

If not, check for the following issues:

• Tracks not displaying at all in JBrowse -- make sure track IDs inside square brackets are of the form [ tracks.-track ]
• JBrowse track is reflected in the X axis -- make sure that the reverse bedgraph file is orientated the correct way; it should be created with the '-f 0x10' flag (no capitalisation).
• JBrowse track only shows one direction -- make sure that the reverse bedgraph file has negative values, and re-generate the bigwig file