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Forum: Sample Prep / Library Generation 07-17-2018, 05:55 AM
Replies: 6
Views: 2,174
Posted By emily202
Of course - makes sense! I think I'm overthinking...

Of course - makes sense! I think I'm overthinking this one :p

Thanks for your input!
Forum: Sample Prep / Library Generation 07-17-2018, 05:35 AM
Replies: 6
Views: 2,174
Posted By emily202
Should I still use a host depletion and bacterial...

Should I still use a host depletion and bacterial enrichment method prior to reverse transcription if I only want to target bacteria for sequencing? Im concerned that the sequencing reads will...
Forum: Sample Prep / Library Generation 07-17-2018, 05:11 AM
Replies: 6
Views: 2,174
Posted By emily202
Thanks for your reply. Yes, RNA-seq can...

Thanks for your reply.

Yes, RNA-seq can provide what's transcriptionally active at the time of sampling. I'm wondering if there is an RNA workflow similar to 16S metagenomic sequencing for...
Forum: Sample Prep / Library Generation 07-12-2018, 11:25 AM
Replies: 6
Views: 2,174
Posted By emily202
Question 16S sequencing from total RNA?

I'm working with a researcher that wants to identify the bacterial communities present in human dental pulp tissue, blood, and saliva from isolated RNA. What is the advantage (or disadvantage) from...
Forum: RNA Sequencing 09-22-2017, 07:04 AM
Replies: 15
Views: 4,988
Posted By emily202
Yes, I plan to prepare all samples using the same...

Yes, I plan to prepare all samples using the same library prep methods.

Thanks very much for your feedback and helpful suggestions.
Forum: RNA Sequencing 09-21-2017, 06:16 AM
Replies: 15
Views: 4,988
Posted By emily202
Thanks, jteeee2. I will definitely try this...

Thanks, jteeee2. I will definitely try this method with a lower SPRI clean-up ratio, as suggested by nucacidhunter. KAPA has always been a reliable NGS resource.
Forum: RNA Sequencing 09-21-2017, 04:41 AM
Replies: 15
Views: 4,988
Posted By emily202
Should some form of fragmentation be performed...

Should some form of fragmentation be performed then? Does no fragmentation introduce bias by not capturing the larger mRNAs in the sample?
Forum: RNA Sequencing 09-21-2017, 03:28 AM
Replies: 15
Views: 4,988
Posted By emily202
Given that the TruSeq kit is only capable of...

Given that the TruSeq kit is only capable of producing up to 200 bp inserts, and I intend to do a 2 x150 bp run, overlap is obviously unavoidable. I do not want to introduce bias in the data analysis...
Forum: RNA Sequencing 09-20-2017, 05:21 PM
Replies: 15
Views: 4,988
Posted By emily202
Yes, annealing/extension is set to 45 sec. I...

Yes, annealing/extension is set to 45 sec.

I was under the assumption that final library insert size was dependent on fragmentation time and post-ligation size selection. Perhaps that is my...
Forum: RNA Sequencing 09-20-2017, 08:07 AM
Replies: 15
Views: 4,988
Posted By emily202
Thanks for the input. I'm using the KAPA library...

Thanks for the input. I'm using the KAPA library quantification kit for qPCR.

I will try altering fragmentation time to 2 min at 94C and doing a double-sided SPRI size selection for 350-500 bp...
Forum: RNA Sequencing 09-19-2017, 03:19 PM
Replies: 15
Views: 4,988
Posted By emily202
Thanks, nucacidhunter, I'm also concerned with...

Thanks, nucacidhunter, I'm also concerned with reading into the adapter or through the adapter into the flow cell and crashing the run; however, we have generated libraries using the "0 minute"...
Forum: RNA Sequencing 09-19-2017, 12:26 PM
Replies: 15
Views: 4,988
Posted By emily202
Question RNA-seq of long RNAs (paired-end, stranded) -- fragmentation time

I'm using the TruSeq Stranded Total RNA library preparation kit to generate libraries for 2x150 bp paired-end sequencing. Illumina's fragmentation protocol recommends 94C for 8 minutes for intact,...
Forum: Bioinformatics 03-14-2017, 08:11 AM
Replies: 0
Views: 1,702
Posted By emily202
Question Extracting HLA-E allele sequences from BAM for gene editing

I have WGS data (2x100bp) that has been aligned to hg19. Using HLA-VBSeq (http://www.biomedcentral.com/qc/1471-2164/16/S2/S7), I HLA typed the data, and now the researcher I am working with is...
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