PoolSeq vs individual short-read polishing comparison (Pieridae)

Knowledge graph centered on PoolSeq vs individual short-read polishing comparison (Pieridae) with 34 nodes and 115 connections. Top connected: Gothic, Pekania pennanti, Colorado East River Valley, Lepidoptera, Mayflies.

Method synopsis

Comparative analysis of genome assembly improvement using pooled vs individual Illumina short reads for polishing long-read assemblies, assessed using BUSCO completeness metrics.

Synthesized from method descriptions across 1 paper using this protocol.

Procedure as described in the canonical source

Steps below were extracted from the paper that introduces this protocol — The genome of the Margined White butterfly (Pieris macdunnoughii): sex chromosome insights and the power of polishing with PoolSeq data (2021), Genome Biology and Evolution. Implementations in other papers (listed below) may differ.

1. Illumina sequencing preparation
   Two sets of Illumina short-read data were produced. Individual sample from Upper East River Valley female collected in 2015. PoolSeq from 18 adult P. macdunnoughii females reared in lab in 2005, originating from Upper East River Valley but likely more inbred. DNA extraction per individual using commercial kit for robotic extraction, with RNAse A treatment. DNA quantified using Qubit 2.0 fluorometer and nanodrop, run on 2% agarose gel with GelRed staining. Equal amounts pooled for library preparation.
   Quantities: 18 females for pooled sequencing, individual extraction amounts not specifiedDuration: Lab rearing in 2005, collection in 2015Conditions: Laboratory conditions
   Equipment: cell and tissue DNA kit, KingFisher Duo Prime purifier (ThermoFisher Scientific), Qubit 2.0 fluorometer (ThermoFisher Scientific), nanodrop (ThermoFisher Scientific), 2% agarose gel, GelRed staining
2. Illumina library preparation and sequencing
   Library preparation and sequencing (Illumina HiSeq) performed by SciLifeLab (Stockholm, Sweden), using 150-bp paired-end reads with 350 bp insert size.
   Quantities: 150-bp paired-end reads, 350 bp insert sizeDuration: Single sequencing runConditions: Commercial sequencing facility
   Equipment: Illumina HiSeq platform
3. Short-read polishing
   Polished the final unpolished genome with each set of mapped Illumina short reads using Pilon (v.1.23). Used SeqKit to subsample 1.60x10^8 of the individual sample reads to make coverage comparable to PoolSeq data. Mapped reads to v0.08 draft assembly with NextGenMap and assessed mapped reads using GoLeft and Qualimap.

Typical Equipment

• Illumina HiSeq platform
• Pilon (v.1.23)
• SeqKit
• NextGenMap

Output Measurements

• BUSCO scores
• assembly completeness
• ortholog hit ratios

Papers Using This Protocol (1)

Species Studied With This Protocol

Places Where Used