Jan 23, 2025

Public workspaceComparative Evaluation of Individual and Pooled Sequencing for Population Genomics Assessment

DOI
dx.doi.org/10.17504/protocols.io.5jyl8dkr7g2w/v1
  • 1CURE-UdelaR;
  • 2INIA;
  • 3CIMMYT;
  • 4Fagro and CURE-UdelaR
Luciana Gillman
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DOI: dx.doi.org/10.17504/protocols.io.5jyl8dkr7g2w/v1
Protocol Citation: Luciana Gillman, Federico Condon, Cesar Petroli, Mercedes Rivas 2025. Comparative Evaluation of Individual and Pooled Sequencing for Population Genomics Assessment. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8dkr7g2w/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 18, 2025
Last Modified: January 23, 2025
Protocol Integer ID: 118676
Funders Acknowledgements:
Luciana Gillman
Grant ID: POS_NAC_2018_1_151772
Luciana Gillman
Grant ID: pasantía 322
Mercedes Rivas
Grant ID: CURE
Federico Condon
Grant ID: INIA
Abstract
This protocol evaluates individual sequencing (ind-seq) and pooled sequencing (pool-seq) datasets, comparing the results obtained with each method. It investigates the correlation between allele frequencies calculated using both approaches to compare their efficacy. Genetic diversity and population structure are also analyzed using both methods. These comparisons explicitly account for the potential effects of sample size, missing data, sequencing depth, and minor allele frequencies.
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Materials
Plant Material: Seeds from your plant or plant.
- Equipment:  
  - Seed germination trays.  
  - NovaDryer-F104 lyophilizer.  
  - Pipettes, centrifuge, and cold storage facilities. 
  - Sequencing platform: Illumina Novaseq 6000.  
  - R-compatible system for bioinformatics analysis.  
- Reagents:  
  - CTAB buffer for DNA extraction.  
  - Ethanol for DNA precipitation.  
  - Restriction enzymes (PstI and MseI).  
  - Barcoded adapters and PCR primers.  
  - Library preparation kits (DArTseq‱ protocol).
 
Seed Germination and Plant Preparation
Seed Germination and Plant Preparation
3w 0d 3h
3w 0d 3h
Germinate seeds under International Seed Testing Association standards.  
1w
Transplant 60 seedlings per accession into trays.  
3h
Allow seedlings to grow until sufficient leaf tissue develops.
2w
Sample Collection
Sample Collection
19h 30m
19h 30m
Individual Samples: Harvest 120 mg of leaf tissue from each of the 60 plants per accession.  
2h
Pooled Samples: Combine tissue from 20 randomly selected plants to create a 20-sample pool (6 mg tissue per plant).
2h
Incrementally add 10 seedlings to form 30-, 40-, 50-, and 60-sample pools, maintaining 4 mg tissue per plant.
15h
Critical
Record individual identities of plants in each pool for comparison with individual sequencing.
30m
Tissue Processing
Tissue Processing
3d
3d
Lyophilize all leaf tissue samples for 72 hours using a NovaDryer-F104. 
2d
Prepare all the sample and paper for shipping.
1d
DNA Extraction and Library Preparation
DNA Extraction and Library Preparation
1d 7h 30m
1d 7h 30m
Extract DNA using the modified CTAB method.  
1d
Digest DNA with PstI and MseI restriction enzymes. 
2h
Ligate barcoded adapters and PCR amplify.  
2h
PCR
3h
Pool PCR products for sequencing. Take into acount sequence each pool at depths of 0.9 Mr and 1.5 Mr and individuals to 0.9 Mr.
30m
Sequencing
Sequencing
1d
1d
Sequence all samples
1d
SNP Calling
SNP Calling
1d
1d
Call SNPs using DArTsoft14 software.  

1d
Data Analysis
Data Analysis
1d 6h
1d 6h
Calculate allele frequencies for individual and pooled samples using R scripts.  
https://github.com/LucianaGillman/ind-pool-seq-hexaploid
3h
Compare SNP metrics (e.g., Representativity, CCC) between datasets.
https://github.com/LucianaGillman/ind-pool-seq-hexaploid/blob/main/calculation_CCC_REP_2.3.3.R
3h
Perform ANOVA to assess effects of sequencing depth, sample size, and minor allele frequency thresholds on SNP metrics.
https://github.com/LucianaGillman/ind-pool-seq-hexaploid/blob/main/ANOVAS_2.7.R
Perform population diversity analyses (e.g., expected heterozygosity, genetic distance) using the dartR and BioR packages.  
https://github.com/LucianaGillman/ind-pool-seq-hexaploid
1d
Protocol references
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