ONT library preparation also allows for multiplexing of samples through the addition of unique 24 bp barcodes to each sample.
After their addition, samples may be pooled together, allowing for
PCR-free multiplexing of up to 24 samples on a single flow cell.
The DNA is again bound to magnetic beads at a 1Â ratio and
cleaned with 80% ethanol washes. The generated sequence can
then be demultiplexed via barcode identification either concurrent
with sequencing or postsequencing.
Ligation of the sequencing adapters is necessary for data acquisition, as these adapters bind to tethers on the ONT flow cell and
act as a guide for the strands of DNA to the pores. After adapter
ligation, the DNA is bound for a final time to magnetic beads at a
0.5Â ratio and washed with long fragment buffer (LFB) provided
by ONT. This change in bead:volume ratio allows for depletion of
fragments 400 bp. LFB, which is an ethanol-free proprietary
reagent, is used as an alternative to an ethanol wash, as the presence
of ethanol at this step could be catastrophic. LFB is also used to
enrich the sample of fragments 3000 bp or longer. If not immediately loaded onto a flow cell, the final adapted libraries may be
stored at 4
C in the short term or at À80
C if storage of the
libraries for longer than 3 months is required.
After completion of library prep, the final library is loaded onto
the sequencing flow cell, which is then inserted into the sequencer.
3.5 Sequencing Read
Preprocessing
and Quality Control
Once the sequencing instrument has been successfully run,
sequencing data are typically output in the FASTQ file format.
3.5.1 Understanding
FASTQ Files
Briefly, FASTQ files (file extension “.fastq”) are text-based files,
which contain strings of DNA bases alongside associated information about that string. These strings of DNA bases are referred to as
reads and represent each fragment of DNA, which was sequenced.
For Illumina paired-end sequencing, the read is a set number of
nucleotides from the flanking region of a library prepped gDNA
fragment, while a Nanopore sequencing read represents an entire
fragment of genomic DNA. In FASTQ files, each read is accompanied by a string of characters of length equal to the number of
DNA bases representing the probability that individual nucleotide
is correctly reported (or “basecalled”); this score is known as the
base’s quality score, “Q score”, or PHRED score. Finally, each
read is accompanied by a unique read ID and a second optional
comment, for a total of 4 lines of text per read. Often FASTQ files
will be “compressed” by the instrument after creation, and FASTQ
files will instead have the extension “.fastq.gz”. Many bioinformatics software programs accept both uncompressed FASTQ files
(.fastq) and compressed FASTQ files (.fastq.gz), but some software
may require decompression of .fastq.gz files prior to their use. On
412
Marco A. Riojas et al.
After their addition, samples may be pooled together, allowing for
PCR-free multiplexing of up to 24 samples on a single flow cell.
The DNA is again bound to magnetic beads at a 1Â ratio and
cleaned with 80% ethanol washes. The generated sequence can
then be demultiplexed via barcode identification either concurrent
with sequencing or postsequencing.
Ligation of the sequencing adapters is necessary for data acquisition, as these adapters bind to tethers on the ONT flow cell and
act as a guide for the strands of DNA to the pores. After adapter
ligation, the DNA is bound for a final time to magnetic beads at a
0.5Â ratio and washed with long fragment buffer (LFB) provided
by ONT. This change in bead:volume ratio allows for depletion of
fragments 400 bp. LFB, which is an ethanol-free proprietary
reagent, is used as an alternative to an ethanol wash, as the presence
of ethanol at this step could be catastrophic. LFB is also used to
enrich the sample of fragments 3000 bp or longer. If not immediately loaded onto a flow cell, the final adapted libraries may be
stored at 4
C in the short term or at À80
C if storage of the
libraries for longer than 3 months is required.
After completion of library prep, the final library is loaded onto
the sequencing flow cell, which is then inserted into the sequencer.
3.5 Sequencing Read
Preprocessing
and Quality Control
Once the sequencing instrument has been successfully run,
sequencing data are typically output in the FASTQ file format.
3.5.1 Understanding
FASTQ Files
Briefly, FASTQ files (file extension “.fastq”) are text-based files,
which contain strings of DNA bases alongside associated information about that string. These strings of DNA bases are referred to as
reads and represent each fragment of DNA, which was sequenced.
For Illumina paired-end sequencing, the read is a set number of
nucleotides from the flanking region of a library prepped gDNA
fragment, while a Nanopore sequencing read represents an entire
fragment of genomic DNA. In FASTQ files, each read is accompanied by a string of characters of length equal to the number of
DNA bases representing the probability that individual nucleotide
is correctly reported (or “basecalled”); this score is known as the
base’s quality score, “Q score”, or PHRED score. Finally, each
read is accompanied by a unique read ID and a second optional
comment, for a total of 4 lines of text per read. Often FASTQ files
will be “compressed” by the instrument after creation, and FASTQ
files will instead have the extension “.fastq.gz”. Many bioinformatics software programs accept both uncompressed FASTQ files
(.fastq) and compressed FASTQ files (.fastq.gz), but some software
may require decompression of .fastq.gz files prior to their use. On
412
Marco A. Riojas et al.