nanopore 微生物文献分析

[anneng]

https://www.biorxiv.org/content/10.1101/2019.12.21.886028v1.full
对牛胃微生物做了分析 对比了nanopore和illumina的两种方式
illumina:
quality control was performed by the same sequencing service using the prinseq-lite program

the forward- and reverse-reads from the sequencing were joined using the FLASH software (Magoc & Salzberg, 2011).

nanopore:
Reads were basecalled and demultiplexed using Guppy
The resulting FASTQ files were used as input of Porechop (Wick R., 2017) for adaptor removal and barcode de-multiplexing

FASTQ files were aligned against the NCBI-nr protein database (Nov. 2017) using DIAMOND v0.9.22 (blastx option) setting the -F option to 15, to consider frame-shift errors in the sequences and the – rangeCulling and –top options set to 10 to scan the whole sequence for alignments with a 10% of the best local bit score

The taxonomic binning of short reads from Illumina was performed using the daa2rma program from MEGAN Community Edition (CE) v6.11. with the option -a2t, to map the reads to the NCBI-taxonomy mapping file containing protein accessions

Relative taxonomic abundances were obtained for each samples and platform representing the number of reads assigned to each taxon.

Relative abundances, alpha (Shannon and Simpson indexes) and beta diversity -using Bray-Curtis dissimilarity-were analyzed using the phyloseq (McMurdie & Holmes, 2013), vegan (Oksanen et al., 2019), and microbiome R (Leo Lahti et al., 2012-2019) packages.

Short reads from the Illumina dataset were assembled using Megahit (Li, Liu, Luo, Sadakane, & Lam, 2015) with the default parameters using the joined forward and reverse reads.

The nanopore reads were assembled using Canu (Koren et al., 2017) setting the options minReadLength = 100, minOverlapLength = 100 as the resulting reads were too short for the default parameters (less than 10,000 bp), and genomeSize = 2.5m, as described in the documentation for metagenome assemblies.

The quality of the assemblies was assessed using Quast v4.0 (Mikheenko, Valin, Prjibelski, Saveliev, & Gurevich, 2016).

Rarefaction curves(稀疏曲线)

alpha diversity indexes

relative abundances