TITLE: Demultiplexing Nanopore reads with LAST
AUTHORS: David Eccles

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol is for a semi-manual method for read demultiplexing, as used after my presentation </span><span style = "font-style:italic;">Sequencing DNA with Linux Cores and Nanopores</span><span> to work out the number of reads captured by different barcodes.</span></div><div class = "text-block">Input: reads as a FASTQ file, barcode sequences as a FASTA file</div><div class = "text-block">Output: reads split into single FASTQ files per target [barcode]</div><div class = "text-block">Note: barcode / adapter sequences are not trimmed by this protocol</div></div>

[STEPS]
?. [Generating Barcode Index]
Prepare a FASTA file containing barcode sequences (see attached FASTA file). To reduce the chance of mismatched adapters, this should only contain the barcode sequences. That restriction means this approach will not work for short reads, where the barcode sequences are very likely to occur within sequences.
?. [Generating Barcode Index]
Prepare the LAST index for the barcode file. Following Martin Frith's recommendation, the '-uNEAR' seeding scheme is used to slightly increase sensitivity. This will generate seven additional files of the form .XXX:lastdb -uNEAR barcode_base.fa barcode_base.fa
?. [Mapping Reads to Barcodes]
Combine all input reads into a single filepv ../called_all/*.fastq | gzip > reads_all.fastq.gzNote: I'm using the pipe viewer command pv to produce a progress indicator while the command is running. If this command is not available, it can be replaced with cat with no change in function (apart from not showing progess).
?. [Mapping Reads to Barcodes]
Use LAST, ignoring FASTQ quality scores for substitution (-Q 0), using the pre-defined substitution matrix to map the reads. In this example, it is distributed over 10 processing threads (-P 10). Here maf-convert is used to convert to a single line per match, cut retains only the barcode and read IDs, and uniq is used to make sure that multiple same barcodes per read (e.g. for reverse / complement barcodes at each end) will not produce duplicates:lastal -Q 0 -P10 -p bc.mat barcode_base.fa   maf-convert -n tab | cut -f 2,7 | sort | uniq | \  gzip > barcode_assignments.txt.gzFor a more stringent search, lastal can do an overlap match (-T 1) rather than the default local match, which will make sure that only the ends of the barcode are matched (hopefully the entire barcode) when demultiplexing. The downside is that this is more likely to drop reads due to slight mismatches in the barcode portion of the read:lastal -T 1 -Q 0 -P10 -p bc.mat barcode_base.fa   maf-convert -n tab | cut -f 2,7 | sort | uniq | \
  gzip > barcode_assignments.txt.gzStringency can also be altered by adjusting the query letters per random alignment setting (-D , 1e6 by default). Lowering this number will produce more matches, at the expense of more false positive matches:lastal -D 1e5 -Q 0 -P10 -p bc.mat barcode_base.fa   maf-convert -n tab | cut -f 2,7 | sort | uniq | \  gzip > barcode_assignments.txt.gzThe output of this command will be a gzipped tab-separated 2-column file with barcode names in the first column, and read IDs in the second column.
?. [Splitting Read File Per Barcode]
For each discovered barcode, using the appropriate read category assignment file, find the corresponding read IDs, then extract those IDs out of the read FASTQ file. This uses one of my scripts, fastx-fetch.pl, to do this directly from a FASTQ file. The '-lengths' command-line parameter also outputs sequence lengths for each read (see next step):mkdir -p demultiplexedfastx-fetch.pl -lengths -demultiplex barcode_assignments.txt.gz \  -prefix 'demultiplexed/reads'  barcode_counts.txtNote: this demultiplexing code will only by default put reads into a barcode bin if they have a single unique barcode sequence detected. Otherwise, they will be put into a 'BCchim' bin if multiple adapters are detected (i.e. a chimeric read), or a 'BCmiss' bin if no adapters are detected. If these reads should be duplicated and put in one bin per barcode, then the -chimeric option can be added to the command arguments:mkdir -p demultiplexedfastx-fetch.pl -lengths -demultiplex barcode_assignments.txt.gz -chimeric \  -prefix 'demultiplexed/reads'  barcode_counts.txt
?. [[optional] Displaying Read Length Statistics]
The lengths output from the demultiplexing step can be fed into another one of my scripts, length-plot.r, in order to display length-based QC plots:fastx-length demultiplexed/lengths_*.txt.gzAs output, this produces a multi-page PDF file, Sequence_curves.pdf. Here are some examples of the plots that are produced:1. Read Count Frequency Curve2. Called Bases Frequency Curve3. Called Bases Density Curve4. Cumulative Sequenced Bases Curve5. Digital Electrophoresis Plot (relative frequency)5. Digital Electrophoresis Plot (sample-normalised)
?. [Downstream Workflows]
Following on from here, cDNA reads can be oriented in preparation for stranded mapping:Preparing Reads for Stranded Mapping
?. [Generating Barcode Index]
Prepare a substitution matrix for barcode mapping. The default substitution matrix is swayed too much by INDELs in the barcode sequences, so here's one that I've developed using a combination of trial & error and last-train:#last -Q 0#last -a 10#last -A 10#last -b 5#last -B 5#last -S 1# score matrix (query letters = columns, reference letters = rows):       A      C      G      TA      4    -24     -9    -24C    -24      5    -24    -14G     -9    -24      7    -24T    -24    -14    -24      8This matrix has a moderate penalty for opening gaps (i.e. insertions and deletions), and a lower penalty for inserting them. Insertions and deletions are considered to be equally likely in the barcode region. It also has a moderate penalty for A/G transition variants, and a higher penalty for C/T transition variants (but still lower than other substitution penalties).