[Research] for long-read sequencing protocol

1. Benchmarking the MinION: Evaluating long reads for microbial profiling

-workflow

The overall experimental design

- basecalling : 전기 신호를  base로 변환하는 단계이다.

- demultiplexing(ref) : 빠른 시퀀싱을 위해 자르고 바코드를 붙였던 read들을 origin sample을 찾는 과정이다.separate files

- porechop 표준 설정으로 정렬 후

- NanoPack 으로 bascalled 및 역다중화 데이터 품질 평가

- classification : kraken(DustMasked MiniKraken DB 8GB), kraken2(MiniKraken2_v1_8GB), centrifuge(Bacteria, Archaea (compressed)), NanoOK(minimap2)

-Visualization : Krona, R

- validation:

Statistics and additional visualizations were computed with R. We calculated the accuracy of the classification performed by Centrifuge, Kraken and Kraken 2 on each sample the proportion of reads assigned to the known input organism at the genus and species level out of the total number reads given any assignment at that rank. To calculate a corresponding estimate of the accompanying error, the mean absolute error, as well as root mean squared deviation of classified to theoretically present fractions on genus and species level were computed. On read level, precision and recall for genus and species identification were computed for Centrifuge, Kraken and Kraken 2 vs. the results obtained from the NanoOK analysis, with precision being the proportion of reads classified correctly to reads classified and recall being the proportion of reads classified correctly to the reads from the NanoOK dataset, which was used as “ground truth”.

 

2. Oxford Nanopore R10.4 long-read sequencing enables the generation of near-finished bacterial genomes from pure cultures and metagenomes without short-read or reference polishing(link)

- Adapters for Nanopore reads were removed using Porechop v. 0.2.3 (ref. 30)

- and reads with a lower length than 200 bp and a Phred quality score below 7 and 10 for R9.4.1 and R10.4 reads, respectively, were removed using NanoFilt v. 2.6.0 (ref. 31).

 

3. Bacteriophage targeting of gut bacterium attenuates alcoholic liver disease (link)

- the sequence reads were demultiplexed and adapters trimmed from ONT reads using Porechop v.0.2.3

 

4. Complete Genome Sequence of an Aeromonas rivuli Strain Isolated from Ready-to-Eat Food (link)

- A quality check using NanoStat v1.5.0 (6) revealed 12,553 reads, with a read N50 value of 14,345 bp and a mean read quality score of 10.7