npGraphServer - gRPC server to run selective sequencing aiming at complete assembly

Introduction

ReadUntil protocol for targeted sequencing requires cricial real-time operations on the streaming output of Nanopore devices. Here, we implemented npGraphServer that provides gRPC interface to invoke from ReadUntil (now ReadFish) python code.

Documentation

For detail options of the commandline interface:

java -cp target/assembly-x.x.x.jar org.rtassembly.NPGraphServerCmd -h

Usage: 
Options:
  --port=i        Port number for which the server will listen to.
                  (default='2105')
  --si=s          Name of the short-read assembly file.
                  (default='')
  --sf=s          Format of the assembly input file. Accepted format are FASTG, GFA
                  (default='')
  --output=s      Output folder for temporary files and the final assembly npgraph_assembly.fasta
                  (default='/tmp/')
  --sb=s          Name of the metaBAT file for binning information (experimental).
                  (default='')
  --overwrite     Whether to overwrite or reuse the intermediate file
                  (default='true')
  --sp            Whether to use SPAdes contigs.paths for bridging.
                  (default='false')
  --qual=i        Minimum quality of alignment to considered
                  (default='10')
  --mincov=i      Minimum number of reads spanning a confident bridge
                  (default='3')
  --maxcov=i      Cut-off number of reads spanning a confident bridge
                  (default='20')
  --depth=i       Limit depth for searching path between 2 neighbors
                  (default='300')
  --anchor=i      Lowerbound of an anchor contig's length.
                  (default='1000')
  --unique=i      Lowerbound of a unique contig's length.
                  (default='10000')
  --expect=i      The readuntil protocol will expect a read to span this length for an unblock/proceed decision.
                  (default='10000')
  --time=i        Time interval to considered for real-time assembly.
                  (default='10')
  --read=i        Read interval to considered for real-time assembly.
                  (default='50')
  --gui           Whether using GUI or not.
                  (default='false')
  --keep          Whether to keep extremely-low-coveraged contigs.
                  (default='false')
  --help          Display this usage and exit
                  (default='false')

The provided functionality is similar to npGraph except no consensus is supported since MSA is slow for this time-critical server. Also PAF is the only alignment format used for rRPC communications since its light-weighted property.

How to

To use the pipeline, first you need to familiarize yourself with ReadFish. A slight modification of this repo to operate with npGraphServer is here. So after install original ReadUntil code, you'll have to update (from the virtualenv) by

pip install -U git+https://github.com/hsnguyen/ru@master

After finishing all installation, you can start to test running selective sequencing with npGraphServer.Beside the Guppy basecall server, you'll need to start a npGraphServer as well for the run.

java -cp target/assembly-x.x.x.jar org.rtassembly.NPGraphServerCmd -si assembly_graph.[fastg|gfa] -output output/ -read 1 -expect 20000 -gui

Follow the procedure similar to ReadFish but using the npgraph_selection.toml instead.

Reference