Oct 30, 2022
Version 7
Preparing Reads for Stranded Mapping V.7
- 1Malaghan Institute of Medical Research (NZ)
Protocol Citation: David A Eccles 2022. Preparing Reads for Stranded Mapping. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvon2zzl4o/v7Version created by David A Eccles
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 30, 2022
Last Modified: October 30, 2022
Protocol Integer ID: 72044
Keywords: long reads, nanopore, strand-specific, sequencing, RNASeq
Abstract
This protocol is for preparing long reads for stranded mapping, as an intermediate step for additional protocols:
- Aligning strand-oriented sequences to a transcriptome for transcript / gene counting
- Aligning strand-oriented sequences to a genome for confirmatory QC
Input(s): demultiplexed fastq files (see protocol Demultiplexing Nanopore reads with LAST), adapter file (containing strand-sensitive adapter sequences)
Output(s): oriented read files, as gzipped fastq files
Barcode Demultiplexing
Barcode Demultiplexing
I usually inspect the barcode_counts.txt file, and delete any barcodes that I'm not interested in:
cp barcode_counts.txt barcode_counts.orig.txt
nano barcode_counts.txt
If demultiplexing has been done correctly, then the following command should produce output without errors:
for bc in $(awk '{print $2}' barcode_counts.txt);
do ls demultiplexed/reads_${bc}.fq.gz;
done
Example output:
demultiplexed/reads_BC03.fq.gz
demultiplexed/reads_BC04.fq.gz
demultiplexed/reads_BC05.fq.gz
demultiplexed/reads_BC06.fq.gz
demultiplexed/reads_BC07.fq.gz
demultiplexed/reads_BC08.fq.gz
If the barcode_counts.txt file is missing, the output will look like this:
awk: fatal: cannot open file `barcode_counts.txt' for reading (No such file or directory)
If one or more of the barcode-demultiplexed files are missing, the output will look something like this:
demultiplexed/reads_BC03.fq.gz
demultiplexed/reads_BC04.fq.gz
demultiplexed/reads_BC05.fq.gz
ls: cannot access 'demultiplexed/reads_BC06.fq.gz': No such file or directory
ls: cannot access 'demultiplexed/reads_BC07.fq.gz': No such file or directory
demultiplexed/reads_BC08.fq.gz
Index Preparation
Index Preparation
Prepare and index a FASTA file containing adapter sequences (see attached FASTA file).
adapter_seqs.fa Create a shell variable containing this file name:
adapterFile="adapter_seqs.fa"
Prepare the LAST index for the adapter file. Newer versions of LAST (v1409+) include a new seeding scheme, '-uRY4' [and other related RYX schemes], which improves mapping accuracy and reduces polyA matches; low-complexity regions are also converted to lower case with '-R01'. This will generate seven additional files of the form <index name>.XXX:
lastdb -uRY4 -R01 ${adapterFile} ${adapterFile}
Prepare a substitution matrix for adapter mapping. Mapping seems to work better when Q values are included:
#last -Q 1
#last -t4.49549
#last -a 46
#last -A 46
#last -b 3
#last -B 3
#last -S 1
# score matrix (query letters = columns, reference letters = rows):
A C G T
A 6 -34 -33 -35
C -34 6 -33 -34
G -33 -33 7 -34
T -35 -34 -34 6
adapter.mat A matrix like this can be generated by the following command, which runs last-train on the first 10,000 reads from the full read dataset:
last-train -Q 1 -P 10 ${adapterFile} <(zcat reads_all.fastq.gz | head -n 40000) | tail -n 13
[note: it is also fine to use the same matrix as used for demultiplexing]
Orienting Reads
Orienting Reads
Use LAST in split mode, using the pre-defined substitution matrix to map the reads. In this example, it is distributed over 10 processing threads (-P 10). In this case it's important that the direction of mapping is also recorded, so the cut command selects three fields (query name [7], target name [2], mapping direction [10]):
for bc in $(awk '{print $2}' barcode_counts.txt);
do echo "** ${bc} **";
lastal --split -P10 -p adapter.mat ${adapterFile} <(pv demultiplexed/reads_${bc}.fq.gz) | \
maf-convert -n tab | cut -f 2,7,10 | uniq | \
gzip > demultiplexed/adapter_assignments_${bc}.txt.gz
done
The adapter assignments are filtered through uniq in order to catch (and exclude) any reads with the strand-switch primer matching multiple times. To unpack the uniq pipe a little bit more, it skips the first field (adapter name), then matches up to 36 characters, retaining only lines that don't match any others. This catches a few more chimeric reads that were missed by the unique barcode filter in the previous protocol.
Reads are filtered into two groups (and one group-by-omission) based on the mapped direction of the strand-switch primer, then reverse-complemented (if necessary) to match the orientation of the original RNA strand. I use my fastx-fetch.pl and fastx-rc.pl scripts for this.
fastx-fetch.pl fastx-rc.pl mkdir -p oriented
for bc in $(awk '{print $2}' barcode_counts.txt);
do echo "** ${bc} **";
fastx-fetch.pl -i <(zgrep '^SSP' demultiplexed/adapter_assignments_${bc}.txt.gz | \
sort | uniq -f 1 -w 36 -u | \
awk '{if($3 == "+"){print $2}}') <(pv demultiplexed/reads_${bc}.fq.gz) | \
gzip > oriented/${bc}_reads_fwd.fq.gz
fastx-fetch.pl -i <(zgrep '^SSP' demultiplexed/adapter_assignments_${bc}.txt.gz | \
sort | uniq -f 1 -w 36 -u | \
awk '{if($3 == "-"){print $2}}') <(pv demultiplexed/reads_${bc}.fq.gz) | \
fastx-rc.pl | gzip > oriented/${bc}_reads_rev.fq.gz
done
Forward and reverse-oriented sequences are combined together to form a single group of RNA-oriented reads.
for bc in $(awk '{print $2}' barcode_counts.txt);
do echo "** ${bc} **";
pv oriented/${bc}_reads_fwd.fq.gz oriented/${bc}_reads_rev.fq.gz | \
zcat | gzip > oriented/${bc}_reads_dirAdjusted.fq.gz
done
Downstream Workflows
Downstream Workflows
Following on from here, the oriented reads can be mapped to a genome (e.g. for visual confirmation of mapping), or to a transcriptome (e.g. for read counting):