Fall 2019: Investigating the Microbial Communities in Mortality Composts
Fall 2019: Investigating the Microbial Communities in Mortality Composts
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Introduction to questions and methods
BIT 477/577 Fall 2019 Students and Carlos Goller
Learning Objectives
- Define next-generation sequencing (NGS).
- Define metagenomics.
- List two applications of metagenomics in health, industry, or medicine.
Next-generation or High-throughput Sequencing
- Define next-generation sequencing (NGS) or high-throughput sequencing. Massively parallel sequencing by synthesis (non-Sanger-based).
- Examples: Illumina MiSeq is “2nd generation,” Nanopore (https://nanoporetech.com/) is “3rd generation” PacBio sequel (https://www.pacb.com/).
- Illumina: Sequencing by synthesis, building DNA model and cataloging sequence by fluorescence
- General Steps/ Method
- PCR desired gene (optional)
- Tag PCR products with bar codes and tags
- Both forward and reverse reads are obtained but forward usually has greater quality
- This is “paired-end” sequencing. Illumina also does “single-read” which is only one direction. Paired-end allows for longer amplicons to be sequenced
- Generate library based on DNA or PCR amplicons
- Library in sequencing refers to a collection of DNA fragments that may be from a chromosome or genomes that have been sheared or digested to a certain size range.
- Sequence on MiSeq
- Analyze via software
- Pitfalls/Limitations:
- Read length
- Read 2 has less quality (Q scores). See this for Illumina Q scores: https://www.illumina.com/documents/products/technotes/technote_Q-Scores.pdf
- Storage and utilization of great amount of generated data
- Expensive (run ~$2,000, reagents and barcodes $900, QIAGEN Smart control $100)’
- Benefits/Advantages
- Lots of reads
- Can generate sequence data from multiple species in a given sample
- Easy to use
- Fast results
- Barcoded library to separate pooled samples using bioinformatics as necessary
- Previous methods
- Capillary required individual bacteria to be grown and cultures, thus individually sequenced.
- Sanger Sequencing: terminally tagged nucleotides
- Required primers
- We used “Phased primers” for QIAseq 16S/ITS (for higher diversity in samples)
- General Steps/ Method
- Nanopore: Still massively parallel, uses many pores to read each strand where each NT disrupts the ionic current and leaves a trace signature
- Trace files: measures ionic disruptions to determine base present.
- Read speed: 400bp/s
- Sequence bins: add the sample to beads to fragment DNA then add adaptor (from Nanopore) that enables barcode identification to separate samples by compost pile.
- General Steps/ Method
- Pitfalls/Limitations:
- Quality is less/ questionable (Q~ 11)
- Storage and utilization of great amount of generated data
- Benefits
- Read length (longest 1mil bp)
- Generates quickly across long strands
- Completely mobile
- Cheap(er) equipment ($900 flowcell and $500 reagents)
- Shotgun sequencing
- Compare/Contrast w/ Illumina
- Similar:
- Massively parallel
- Different:
- Measuring disruption in ionic