detect

DNAscent detect is a DNAscent subprogram that analyses each nanopore-sequenced read and, at each thymidine position, assigns the probability that the base is really BrdU and EdU.

Usage

To run DNAscent detect, do:
   DNAscent detect -b /path/to/alignment.bam -r /path/to/reference.fasta -i /path/to/index.dnascent -o /path/to/output.detect
Required arguments are:
  -b,--bam                  path to alignment BAM file,
  -r,--reference            path to genome reference in fasta format,
  -i,--index                path to DNAscent index,
  -o,--output               path to output file that will be generated.
Optional arguments are:
  -t,--threads              number of threads (default is 1 thread),
  --GPU                      use the GPU device indicated for prediction (default is CPU),
  -q,--quality              minimum mapping quality (default is 20),
  -l,--length               minimum read length in bp (default is 1000).

The main input of DNAscent detect is an alignment (bam file) between the sequence fastq from Guppy and the organism’s reference genome. This bam file should be sorted using samtools sort and indexed using samtools index so that there is a .bam.bai file in the same directory as the bam file. (Please see the example in Workflow for details on how to do this.) The full path to the reference genome used in the alignment should be passed using the -r flag, and the index required by the -i flag is the file created using DNAscent index (see index).

The number of threads is specified using the -t flag. DNAscent detect multithreads quite well by analysing a separate read on each thread, so multithreading is recommended. By default, the signal alignments and ResNet predictions are run on CPUs. If a CUDA-compatible GPU device is specified using the --GPU flag, then the signal alignments will be run on CPUs using the threads specified with -t and the ResNet BrdU and EdU predictions will be run on the GPU. Your GPU device number can be found with the command nvidia-smi. GPU use requires that CUDA and cuDNN are set up correctly on your system and that these libraries can be accessed. If they’re not, DNAscent will default back to using CPUs.

It is sometimes useful to only run DNAscent detect on reads that exceed a certain mapping quality or length threshold (as measured by the subsequence of the contig that the read maps to). In order to do this without having to filter the bam file, DNAscent provides the -l and -q flags. Any read in the bam file with a reference length lower than the value specificed with -l or a mapping quality lower than the value specified with -q will be ignored.

Before calling BrdU and EdU in a read, DNAscent detect must first perform a fast event alignment (see https://www.biorxiv.org/content/10.1101/130633v2 for more details). Quality control checks are performed on these alignments, and if they’re not passed, then the read fails and is ignored. Hence, the number of reads in the output file will be slightly lower than the number of input reads. Typical failure rates are about 5-10%, although this will vary slightly depending on the read length, the analogue substitution rate, and the genome sequenced.

Output

DNAscent detect will produce a single human-readable output file with the name and location that you specified using the -o flag. To aid organisation and reproducibility, each detect file starts with a short header. The start of each header line is always a hash (#) character, and it specifies the input files and settings used, as well as the version and commit of DNAscent that produced the file. An example is as follows:

#Alignment /path/to/alignment.bam
#Genome /path/to/reference.fasta
#Index /path/to/index.dnascent
#Threads 1
#Compute CPU
#Mode CNN
#MappingQuality 20
#MappingLength 5000
#SystemStartTime 09/06/2022 12:45:29
#Software /path/to/DNAscent
#Version 3.0.0
#Commit 4cf80a7b89bdf510a91b54572f8f94d3daf9b167

You can easily access the header of any .detect file with head -12 /path/to/output.detect or, alternatively, grep '#' /path/to/output.detect.

Below the header is data for each read. Note that everything in this output file orients to the reference genome in the 5’ –> 3’ direction. Each read starts with a line in the format:

>readID contig mappingStart mappingEnd strand

These lines always begin with a greater-than (>) character. Therefore, an easy way to count the number of reads in the file is grep '>' detect.out | wc -l. The fields are:

  • readID is a unique hexadecimal string assigned to each read by the Oxford Nanopore software,
  • the read mapped between mappingStart and mappingEnd on contig,
  • strand either takes the value fwd, indicating that the read mapped to the forward strand, or rev indicating that the read mapped to the reverse complement strand.

The following shows an example for a read that to the reverse strand between 48490 and 53033 on chromosome 1.

>0d64a203-81b5-4b6c-aa2f-67b20969a509 1 48490 53033 rev

Below these “start of read” lines, each line corresponds to the position of a thymidine in that read. There are four tab-separated columns:

  • the coordinate on the reference,
  • probability that the thymidine is actually EdU,
  • probability that the thymidine is actually BrdU,
  • 6mer on the reference.

Consider the following examples:

>a4ea2872-9cb6-4218-afad-905f79204eb1 14 992440 996846 rev
992448  0.125751        0.131483        ATCTTA
992450  0.082488        0.078428        CTTATA
992451  0.070718        0.050604        TTATAA
992453  0.062216        0.047409        ATAACA
992456  0.056369        0.042582        ACATTA
992457  0.046755        0.038603        CATTAA
992459  0.056535        0.041545        TTAATA

Here, we’re looking at the sequence ATCTTATAACATTAATA on the reference genome. Because this read maps to the reverse complement, a call is made at every A (instead of T) on the reference. The low probabilities of BrdU and EdU indicate the shown region of this particular molecule is unlikely to have analouge incorporated in it.

If instead we looked at a read that mapped to the forward strand, an example would be:

>d1e97c0f-5de7-4249-a426-30d5b4334106 2 748326 761207 fwd
748327  0.066334        0.084699        TTTAGA
748328  0.045942        0.152147        TTAGAA
748329  0.040509        0.187831        TAGAAA
748336  0.028645        0.278245        TCGGAC
748352  0.021041        0.922350        TCGAAT
748357  0.017188        0.932314        TGTAAT
748359  0.016415        0.921368        TAATAT

Here, we have a few genomic positions with high probability BrdU calls, indicating that the shown region of this molecule may be BrdU-substituted.