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rRNA maturation

Basecalling and demultiplexing of raw reads

Table of contents
  1. Basecalling of raw reads using guppy_basecaller
  2. Demultiplexing of basecalled reads using guppy_barcoder
  • For better comparability we re-basecalled the FAST5 file from all runs using the same version on guppy on a Mk1C (ont-guppy-for-mk1c v4.3.4)
  • Once the Mk1C is set up correctly and connected to the internet, updates are displayed automatically and can be completed by click
  • Other software downloads are available from the ONT downloads page

Basecalling of raw reads using guppy_basecaller

  • After sequencing (and despite live-basecalling) all datasets in the raw_FAST5 folder were re-basecalled using guppy (ont-guppy-for-mk1c v4.3.4) in high-accuracy mode (rna_r9.4.1_70bps_hac.cfg, dna_r9.4.1_450bps_hac.cfg) without quality filtering
  • The output files in FASTQ format were written to the basecalled folder.

Note: Mk1C

  • We (and others) noticed the apparently acces via GUI is not the fastest way to perform basecalling
  • In case you want to optimise GPU-accelerated basecalling on the Mk1C (settings for NVIDIA Jetson TX2) using have a look at Miles Benton`s GitHub

According this his suggestions (which really speed up the process), we performed basecalling like this (using ssh access to the Mk1C):

# files
input=microbepore/data/raw_FAST5/run_id # add run id
output_DRS=microbepore/data/FASTQ/normal/run_id # add run id
output_cDNA=microbepore/data/basecalled/run_id # add run id

# Basecalling of DRS files
guppy_basecaller \
--input_path ${input} \ # input path
--save_path ${output_DRS} \ # output path
-c rna_r9.4.1_70bps_hac.cfg  \ # config file: high accuracy RNA
--calib_detect \ # detect calibration spike-in
--reverse_sequence true \ # reverse since sequenced 3´-->5´
--u_substitution true \ # replace U´s with T´s
--compress_fastq \ # compress output
--fast5_out \ # output FAST5
--recursive \ # look for FAST5 recursively in path
--progress_stats_frequency 60 \ # output progress every minute
--chunks_per_runner 256 \ # options for Mk1C
--gpu_runners_per_device 4 \ # options for Mk1C
--num_callers 1 \ # options for Mk1C
-x auto # options for Mk1C

# Basecalling of cDNA files 
guppy_basecaller \
--input_path ${input} \
--save_path ${output_cDNA} \
-c dna_r9.4.1_450bps_hac.cfg \ # config file: high accuracy cDNA 
--compress_fastq \
--fast5_out \
--recursive \
--progress_stats_frequency 60 \
--chunks_per_runner 256 \
--gpu_runners_per_device 4 \
--num_callers 1 \
-x auto

Note:

  • DRS & (PCR-)cDNA runs require different options.
  • Config file selection based on selected accuracy, flowcell version, library preparation kit are listed with guppy_basecaller --print_workflows

Since basecalling is probably taking quite a while, but you want to keep the process running without interruption you can use the nohup command. You can use it like nohup guppy_basecaller ..... &. This starts basecalling using guppy and puts to process in the background (that´s what the & does). Now, you can log out from the Mk1C and the process keeps running. To monitor the process you can check the shell output that is written to nohup.out.

With the selected options guppy produces fast5_pass, fast5_fail, fastq, summary and report files that are written to the FASTQ folder. FASTQ are not grouped in pass and fail groups since --min_qscore is not enabled. Multiple FASTQs can be merged using cat microbepore/data/basecalled/run_id/*.fastq > microbepore/data/basecalled/run_id/run_id.fastq.

Sequencing summary files are also written to the FASTQ folder and are used during the quality control of the runs and reads. For better viewing they can be moved to the summary folder using mv microbepore/data/FASTQ/run_id/sequencing_summary.txt microbepore/data/summary/run_id.txt

Demultiplexing of basecalled reads using guppy_barcoder

Next, multiplexed cDNA libraries are demultiplexed in a separate step using guppy_barcoder.

# files
input=microbepore/data/basecalled/run_id # add run id
output=microbepore/data/FASTQ/normal/run_id # add run id

# Demultiplexing of (PCR-)cDNA files
guppy_barcoder \
--input_path ${input} \
--save_path ${output} \
--config configuration.cfg \
--barcode_kits SQK-PCB109 \
--progress_stats_frequency 60

Multiple FASTQs are written to the FASTQ folder and can be merged with e.g. cat microbepore/data/FASTQ/run_id/barcode01/*.fastq > microbepore/data/FASTQ/run_id/run_id_barcode01.fastq. Barcode summary files are written to the FASTQ folder and can be moved to the barcode folder.