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ZYMO RESEARCH CORP.
Phone: (949) 679-1190 ▪ Toll Free: (888) 882-9682 ▪ Fax: (949) 266-9452 ▪ [email protected] ▪ www.zymoresearch.com
Appendix D
Troubleshooting:
For
Technical Assistance
, please contact 1-888-882-9682 or E-mail [email protected].
Problem
Possible Causes and Suggested Solutions
•
Clean workspace, centrifuge, and pipettes with 10% bleach to
routinely to avoid contamination.
•
Use of kit in exposed environment without proper filtration.
Check pipettes, pipette tips, microcentrifuge tubes, workspace,
etc. for contamination.
Background Contamination
•
Make sure bags of columns and buffer bottles are properly
sealed for storage. Use of these outside a clean room or hood
can result in contamination.
A)
Use ZymoBIOMICS
™
Microbial Standards for assessing GC-Bias
in Shotgun Metagenomics
B)
Perfect for tracking PCR Chimera in 16S
rRNA Gene Sequencing
Figure 9.
A)
Library preparation for shotgun metagenomic sequencing was performed in two different ways: one by supplier I and one by an in-house method. Shotgun
sequencing was performed on Illumina
®
MiSeq
™
with paired-end sequencing (2 x 150 bp). Raw reads were mapped to the 10 microbial genomes to evaluate the
potential effect of GC content on sequencing coverage. Normalized coverage was calculated by normalization by the average sequencing coverage of each
genome
B)
PCR chimera increases with PCR cycle number in the library preparation process of 16S rRNA gene targeted sequencing. 20 ng ZymoBIOMICS
™
Microbial
Community Standard was used a template. The PCR reaction was performed with ZymoBIOMICS
™
PCR Premix and with primers that target v3-4 region of 16S
rRNA gene. Chimera rate in percentage was determined with Uchime and using the 16S rRNA gene of the 8 bacterial strains in the standard as reference PCR.
chimera sequences were defined as sequences resulted from the recombination/hybridization of different template sequences.
