Libraries were prepared according to the COVID-19 ARTIC v3 Illumina Library Construction and Sequencing Protocol V.5 [19 ], using the NEBNext Ultra DNA Library Preparation Kit II (New England Biolabs Inc., Ipswich, MA, USA). Libraries were quantified (Qubit DNA BR, Thermo Scientific, Waltham, MA, USA), normalized, and pooled, and sequencing was performed using an Illumina MiSeq 100 (Illumina, San Diego, CA, USA).
Miseq 100
Manufactured by Illumina
Sourced in United States
The MiSeq 100 is a desktop sequencing system designed for a wide range of applications, including targeted sequencing, small genome sequencing, and microbial identification. It utilizes proprietary sequencing-by-synthesis technology to generate high-quality sequencing data.
Automatically generated - may contain errors
3 protocols using miseq 100
1
COVID-19 ARTIC v3 Illumina Sequencing
2
Infant Gut Microbiome Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Infant stools were collected from diapers and aliquoted within 24 hours into sterile tubes and stored at −80° C. DNA extraction was then conducted using Zymo DNA extraction kits (Tustin, CA, USA) on thawed samples, followed by quantity and purity assessment using OD260/280 nanodrop measurement (19 (link)). Fusion primers were used to obtain 16S ribosomal RNA (rRNA) V4-V5 amplicons. One of eight five-nucleotide long forward primers was used to connect the Illumina-specific bridge and sequencing primer regions with the 16S region, whereas the reverse primer was simply one of 12 Illumina indices.
16S rRNA amplification of the V4-V5 hypervariable regions was conducted at the Marine Biological Laboratory in Woods Hole, Massachusetts, USA for microbial profiling using both forward and reverse primers (20 (link), 21 (link)). Due to the use of both forward and reverse primers, 96 samples were multiplexed per lane. PCR was then conducted using triplicate samples of 33 μL, and a combination of 1.0 U Platinum Taq Hi-Fidelity Polymerase (Life Technologies Carlsbad, CA, USA), 1X Hi-Fidelity buffer, 200 μM dNTP3 PurePeak DNA polymerase mix (Pierce Nucleic Acid Technologies Milwaukee, WI, USA), 1.5 mM MgSO4 and 0.2 μM of each primer. Lastly, qPCR was used to quantify each library pool before sequencing using paired-end Illumina MiSeq 100 cycle runs.
16S rRNA amplification of the V4-V5 hypervariable regions was conducted at the Marine Biological Laboratory in Woods Hole, Massachusetts, USA for microbial profiling using both forward and reverse primers (20 (link), 21 (link)). Due to the use of both forward and reverse primers, 96 samples were multiplexed per lane. PCR was then conducted using triplicate samples of 33 μL, and a combination of 1.0 U Platinum Taq Hi-Fidelity Polymerase (Life Technologies Carlsbad, CA, USA), 1X Hi-Fidelity buffer, 200 μM dNTP3 PurePeak DNA polymerase mix (Pierce Nucleic Acid Technologies Milwaukee, WI, USA), 1.5 mM MgSO4 and 0.2 μM of each primer. Lastly, qPCR was used to quantify each library pool before sequencing using paired-end Illumina MiSeq 100 cycle runs.
3
Infant Gut Microbiome Profiling
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!