Philip Lyons

11 Questions 33 Answers 0 Followers

Questions related from Philip Lyons

Is rarefaction essential when examining 16s amplicons from illumina NGS?

I am examining 16s diversity from intestinal content of fish to look at the microbial diversity in each sample. Looking throught the literature, some papers use rarefaction analysis and some...

24 January 2015 5,269 19 View

When making 16S V4 libraries, what concentration of DNA per sample is pooled after indexing PCR?

Is there an optimum amount per sample in microbiome analysis?

13 January 2015 3,587 1 View

What concentration of DNA is optimal after first step amplicon PCR in illumina 16s sequencing?

I am getting ok bands after first step PCR - measuring 1-2ng/ul, is this enough?

25 December 2014 7,169 7 View

What is the optimum DNA concentration and purity for 16S NGS?

I am extracting DNA from fish gut to characterise the microbial community. I have found that DNA yields are usually low (between 50 and 300ng/ul using nanodrop/qubit). I am curious to know the...

06 November 2014 7,160 5 View

What is an acceptable sequencing depth for microbiome analysis?

I am undertaking 16S amplicon microbiome characterization of fish gut using MiSeq. Just wondering if anybody has any similar experience in figuring out a required sequencing depth to achieve this...

30 October 2014 3,694 7 View

Can anyone help with inhibitex tablets for PCR inhibitor removal?

Does anybody have the Inhibitex tablets (Qiagen) for removal of PCR inhibitors from DNA extraction preps? I have a sampling coming up and really need them. Thank you

15 July 2014 1,363 9 View

Has anyone used the InhibitEx Buffer in new Qiagen Fast DNA Stool Mini Kit?

I have always used Qiagen's DNA Stool Mini Kit for extraction of community DNA from environmental samples. However, the manufacturer has no replaced this kit with "Fast DNA Stool Mini Kit", which...

02 June 2014 7,509 15 View

What is the typical number of reads from a illumina MiSeq 16S microbial community profile from an environmental sample?

I ran 10 samples altogether, yielding a total of 10,289,176 reads before quality filtering. After removal of chimeras and denoising (up to OTU assignment), the total was 4,560,861. I then quality...

27 March 2014 741 9 View

Can analyzing sequence clusters, as opposed to raw reads help to eliminate bias when samples return different numbers of total reads?

I am analyzing microbiome data from a pyroseq run. My analysis returned different numbers of reads for each sample. Can clustering of sequences belonging to different genera help to eliminate any...

13 March 2014 2,633 2 View

When sampling small amounts of intestinal contents (approx. 150mg), is it preferable to perform DNA extractions right away?

Or to immediately snap freeze in liquid N or at -80CIf storage is in option, can the samples be stored in a sterile eppendorf only, or does the eppendorf have to contain a stabilizing buffer...

24 January 2014 9,890 2 View

Sanger Sequencing of a 16S PCR Product?

I sequenced a 16S product, generated from template DNA from an environmental sample. There were only a few "n" base calls in the retrieved sequence from this product, and the sequence showed 100%...

08 January 2014 789 34 View