Library Construction for Next-Generation Sequencing

 

Please note that next-generation sequencing of libraries prepared by the Genomic Diversity Facility (GDF) is usually performed at the BRC Genomics Facility.  Unless other arrangements are requested, DNA sequencing orders will be made by the GDF and sequencing charges will be included in our quotations.

Whole genome sequencing

Library construction, including sample quantification and library QC, is currently available for small insert (300 or 500 bp), PCR-free, Nextera XT (bacterial genomes), Nextera (large genomes), or TruSeq libraries (8 sample minimum). Sample submission.

 

3’RNAseq

The Genomic Diversity Facility now offers high-throughput 3’ RNA seq (48-, 96- or 192-plexes) at a cost to internal (Cornell) investigators of $30-$54 per sample, depending on multiplex level.  This price includes library construction and sequencing on the NextSeq 500 (90 nt, single-end reads).   Sample submission.

3’RNA seq (see diagram below) is the best alternative to microarrays and conventional RNA seq for gene counting, expression and eQTL studies.  High strand specificity allows discovery and quantification of antisense transcripts and overlapping genes.  Because the exact position of the 3’end of the polyA RNA is determined, accurate sequence information is obtained for the 3’UTR.  More reliable gene expression values are obtained because only one cDNA is produced per transcript (length normalization is not required).  Unlike conventional RNA seq, however, 3’RNA seq does not generate information on splice-site junctions/alternative splicing.

 

Custom Genotyping

A new genotyping method, rAmpSeq, based on amplifying unique loci (several thousand per DNA sample) using primers anchored in repetitive DNA (i.e., transposable elements) is currently in development for maize (Zea mays), cassava (Manihot esculenta), sorghum (Sorghum bicolor), and additional species.  Because up to 3,072 samples can be multiplexed in one sequencing lane, this technology is well-suited for large genomic selection projects.  The protocol is now available for maize and cassava (inquire, BRC_Genomic-Diversity@cornell.edu).

Barcoding of purified PCR products for multiplex sequencing of custom amplicons is now available using either Nextera or TruSeq dual indices (inquire, BRC_Genomics@cornell.edu).