Library prep for FFPE samples /Illumina

srath · 01-04-2018, 03:05 PM

I am looking for library prep kits compatible with illumina NextSeq/NovaSeq platform. I have RNA and DNA from FFPE samples and am looking for kits for both RNASeq and exome sequencing.

Could you please guide me on the most cost effective and high quality kit.

I am thinking about using KAPA/ROCHE HYPERPLUS kit for exome sequencing.

I am debating between

TruSeq Stranded Total RNA Library Prep by illimina or TrueSeq RNA ACCESS by illumina for RNASeq.

Q.1. What is the difference between these two kits?
Q.2. I do not have a germline control at the moment. What can I do about that?

Edinburgher · 01-18-2018, 12:07 AM

I am using the RNA access kit. It is better for degraded samples.

lexogen · 03-10-2018, 01:24 PM

Hi, for gene expression analysis please have a look at the QuantSeq kit from Lexogen: https://www.lexogen.com/quantseq-3mrna-sequencing/

Due to it's focus on 3' ends this established method shows excellent gene expression determination perfomance with heavily degraded and FFPE-derived RNA - from USD 19.80 per sample incl. data analysis!

If you have any further questions, please e-mail info@lexogen.com and have a look at Fig. 7 and Fig. 8 of the QuantSeq Application Note.

Lukas Paul
Sr. Manager of Scientific Affairs

Marc_Jones · 04-12-2018, 12:56 AM

Quote:

Originally Posted by Edinburgher

I am using the RNA access kit. It is better for degraded samples.

Edinburgher what multiplexing/pooling strategy do you use?

I would like to pool more than 24 samples...closer to 48 and use a NovaSeq but I don't think it's possible?

GSviral · 04-13-2018, 02:55 AM

Hi Marc,

I can confirm from experience that the TruSeq RNA Access (now RNA Exome) performs really well with degraded FFPE material and generates consistent, high quality libraries from FFPE samples even with low DV200 scores. As far as multiplexing goes the kit has options for a Set A and Set B index kit which gives a total of 24 unique libraries for pooling and sequencing. I don't have any experience in using other index possibilities with the kit (like dual indexing) for a greater amount of uniquely barcoded libraries. It may be worth contacting Illumina to see if a solution exists.

GSviral · 04-13-2018, 03:07 AM

Marc,

A quick look on the product page for the TruSeq RNA Exome kit states that multiplex options are 24 single index or up to 96 combinatorial dual indexes. This seems like it would meet your criteria for your 48 samples multiplexing.

Marc_Jones · 04-13-2018, 05:31 AM

Thanks all

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01-04-2018, 03:05 PM	#1
srath Junior Member Location: phoenix Join Date: Jan 2018 Posts: 1	Library prep for FFPE samples /Illumina I am looking for library prep kits compatible with illumina NextSeq/NovaSeq platform. I have RNA and DNA from FFPE samples and am looking for kits for both RNASeq and exome sequencing. Could you please guide me on the most cost effective and high quality kit. I am thinking about using KAPA/ROCHE HYPERPLUS kit for exome sequencing. I am debating between TruSeq Stranded Total RNA Library Prep by illimina or TrueSeq RNA ACCESS by illumina for RNASeq. Q.1. What is the difference between these two kits? Q.2. I do not have a germline control at the moment. What can I do about that?

03-10-2018, 01:24 PM	#3
lexogen Registered Vendor Location: Vienna, Austria and NH, USA Join Date: Aug 2012 Posts: 49	QuantSeq 3' mRNA-seq lib prep kit for degraded RNA Hi, for gene expression analysis please have a look at the QuantSeq kit from Lexogen: https://www.lexogen.com/quantseq-3mrna-sequencing/ Due to it's focus on 3' ends this established method shows excellent gene expression determination perfomance with heavily degraded and FFPE-derived RNA - from USD 19.80 per sample incl. data analysis! If you have any further questions, please e-mail info@lexogen.com and have a look at Fig. 7 and Fig. 8 of the QuantSeq Application Note. Lukas Paul Sr. Manager of Scientific Affairs

01-18-2018, 12:07 AM	#2
Edinburgher Junior Member Location: San Francisco Join Date: Jan 2018 Posts: 4	I am using the RNA access kit. It is better for degraded samples.

04-13-2018, 02:55 AM	#5
GSviral Member Location: UK Join Date: Dec 2014 Posts: 37	Hi Marc, I can confirm from experience that the TruSeq RNA Access (now RNA Exome) performs really well with degraded FFPE material and generates consistent, high quality libraries from FFPE samples even with low DV200 scores. As far as multiplexing goes the kit has options for a Set A and Set B index kit which gives a total of 24 unique libraries for pooling and sequencing. I don't have any experience in using other index possibilities with the kit (like dual indexing) for a greater amount of uniquely barcoded libraries. It may be worth contacting Illumina to see if a solution exists.

04-13-2018, 03:07 AM	#6
GSviral Member Location: UK Join Date: Dec 2014 Posts: 37	Marc, A quick look on the product page for the TruSeq RNA Exome kit states that multiplex options are 24 single index or up to 96 combinatorial dual indexes. This seems like it would meet your criteria for your 48 samples multiplexing.

04-13-2018, 05:31 AM	#7
Marc_Jones Member Location: UK Join Date: Jun 2017 Posts: 10	Thanks all