Quality Control for Nanopore Data

Presentations Thursday. Give us the 5 minute pitch for your project covering:

• What questions the study is asking?
• What data did they collect?
• Show at least one figure from the paper that aims to answer the main questions.
• Present the flow of analyses/programs used. (A flow chart might be nice)

We will be exploring Nanopore read quality and filtering. This is always an important step with any sequencing data.

Common QC Steps:

• Read length distribution
• Data complexity/kmer counting
• Quality score mapping (usually by read length, but in Nanopore also possible through time with raw data)
• Adapter sequence removal
• Detect duplicates

In the wider world of Bioinformatics the king of QC is fastqc.

However, fastqc has trouble with Nanopore data and ONT specific tools exist.

Tools:

• MinIONQC – Operates directly on the summary files, not the fast5 or fastq. We do not have any of those files yet.
• NanoPlot / NanoStat / NanoFilt
• porechop Adapter removal
• poretools – output some quality stats, also converts to other file types.
• Others??

Example run:

NanoStat --fastq $datapath/SRR5805004.fastq > ns.file

General summary:
Mean read length:               6,089.3
Mean read quality:                 11.6
Median read length:             6,101.0
Median read quality:               11.7
Number of reads:               29,280.0
Read length N50:                7,783.0
Total bases:              178,296,024.0
Number, percentage and megabases of reads above quality cutoffs
>Q5:    29280 (100.0%) 178.3Mb
>Q7:    29280 (100.0%) 178.3Mb
>Q10:   24456 (83.5%) 151.5Mb
>Q12:   12284 (42.0%) 78.3Mb
>Q15:   243 (0.8%) 1.7Mb
Top 5 highest mean basecall quality scores and their read lengths
1:      16.7 (2228)
2:      16.7 (8360)
3:      16.7 (5289)
4:      16.6 (3013)
5:      16.6 (8902)
Top 5 longest reads and their mean basecall quality score
1:      46833 (10.9)
2:      40311 (14.1)
3:      35785 (11.8)
4:      33627 (9.6)
5:      31740 (12.6)

Visualize quality metrics

NanoPlot --fastq $datapath/SRR5805004.fastq --outdir nanoplots

Output page

Filter reads on quality, read length, GC content, and trimming off the start or end of reads (to ensure adapter removal).

cat $datapath/SRR5805004.fastq | NanoFilt -q 10 -l 500 > filt.re
ads.fq

less filt.reads.fq

Always look at your data before deciding on filtering settings. There is no magic setting that will work for every study.

Be aware of all of the options:

NanoFilt -h

porechop -i $datapath/SRR5805004.fastq  -o out.fq #Remove adapters from fastq file, write to out.fq

poretools operates directly on fast5 files that have basecalls.

Can generate a variety of informat:

poretools -h 
#typical command
#poretools command path/to/fast5/experimentfiles

Guppy Basecalling for oryza and snake_venom projects