11 February 2014 6 9K Report

Please suggest mathematical model and how to implement it in MIMO.

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What evalues are recommended for BLASTing fungal sequences?

Hello, I'm using the command line to NCBI BLAST fungal DNA sequences (Table-C1, https://www.ncbi.nlm.nih.gov/books/NBK279684/). The default evalue is 10.0, but one paper I'm following used...

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Grouping read IDs by sample names, and sorting samples into separate fasta files?

Dear colleagues, I pooled together dozens of samples into one library, and sequenced them together. This yielded millions of reads, with the sequencer assigning each unique read-IDs. While the...

08 October 2018 5,513 4 View

What are acceptable thresholds when using BLASTn on fungal DNA sequences?

Hello, I'd like to know your opinions on identifying fungal DNA sequences using the BLASTn with regards to Max Score, Total Score, Query Cover, E-value, and Identity match %? What are common...

06 July 2018 9,896 1 View

Similarity thresholds for fungal OTUs?

Hello, I understand there's disagreement regarding the similarity threshold for fungal OTUs, be it 95%, 97%, or 90-99%. In the literature, anyone of these threshold may be cast as adequate or too...

01 June 2018 3,308 4 View

Ligations: Using DNA ligases and buffers of E. coli, and/or T4?

Hello, I'm following Thermofisher's "Ion 16S Metagenomics Kit User Guide", and I want to ligate Ion P1 Adaptors, and an Ion Xpress Barcode onto dsDNA amplicons for ion torrent sequencing (p. 16,...

29 March 2018 2,018 2 View

Removal of excessive primer dimers: Use multiple sessions of Ampure XP bead purification?

Hello all, Previously after completing my PCRs, their electrophoresis gels indicated all PCRs had primer dimers. I am having difficulty ridding my PCRs of dimers as well as anything 100...

14 December 2017 2,943 4 View

Can I avoid diluting my PCRs to equal molar concentrations before I pool them?

Background: First, I want to sequence fungal DNA collected from samples of over 100 individual trees. Each PCR sample was amplified with an unique barcoded combination of forward and reverse...

12 December 2017 5,083 2 View

Handling PCR inhibitors: Combinations of BSA, Tween-20, MgCl2, DMSO, and/or Betaine? Also GC content of fungal sequences?

Hello all, I am trying to optimize my PCRs to amplify fungal DNA isolated from extracts of mostly plant DNA. I'm using ITS-1F and ITS-2R for my forward and reverse primers, respectively. Past...

29 October 2017 5,299 6 View

How to determine the Tm of barcoded primers?

Hi all, I need help calculating the melting temperature (Tm) of barcoded primers for PCRs. I am using two barcoded primers to separate dozens of samples' DNA sequences. Using the equation...

06 September 2017 2,650 3 View

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