Progress 11/01/02 to 10/31/05
Outputs
4d Progress report. This report serves to document research conducted under a National Research initiative competitive grant from CSREES, USDA. Additional details of research can be found in the report for the parent project 1265-32000-072-00D "Immunological and genetic basis of resistance to parasites of cattle". We have completed construction and sequence characterization of two normalized cDNA libraries (BARC 9BOV & 10BOV) derived from gut tissues undergoing parasitic infection. The libraries were constructed from the following tissues: 1) for 9BOV fundic and pyloric sections of abomasum from the O. ostertagi infected 15 week-old Holstein steers were collected three and six weeks post-infection and 2) for 10 BOV similar temporal collection of proximal jejunum was done for a C. oncophora infected group and combined with Cryptosporidium parvum infected distal ileum collected seven days after infection of two seven- day old male Holstein calves. Normalization
effectively reduced redundancy 130 and 110-fold for 9BOV and 10BOV, respectively. The normalized 9BOV and 10BOV libraries contained approximately 1.1x107 and 2. 1x107 total transformants with average insert length of 1.0 and 1.2 Kb, respectively. A total of 12,672 individual colonies from each library were picked and arrayed into 33 384-well plates prior. These plates were processed in entirety for plasmid-based 5' and 3'-end (bi-directional) sequencing. Public distribution of clones and library copies is being arranged with BACPAC resources. A total of 50,112 sequence trace files were assessed for quality to generate 43,772 sequences with 100 or more high-quality bases (Phred score > 18) after trimming and removal of vector and contaminating bacterial-derived sequences. After removal of 42 EST matching O. ostertagi sequences and C. parvum accessions, the remaining 43,730 EST were deposited into GenBank. Approximately 20% of the EST represents new bovine gene sequences relative to
existing sequence information in GenBank dbEST. Analysis of the EST information is ongoing for a manuscript. However, 20% of the clones from the libraries contain full length open reading frame cDNA inserts, and 2,000 of these have been fully sequenced. EST trace files for the 25,000 3' reads have been distributed to Texas A&M and Michigan State for design of long oligo- nucleotides for microarrays. This information has already added more than 3,000 new and annotated targets to the Michigan State array to expand their version one array above 10,000 targets.
Impacts
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Publications
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Progress 10/01/03 to 09/30/04
Outputs
4. What were the most significant accomplishments this past year? This report serves to document research conducted under a Reimbursable Agreement (NRI Grant) from CSREES, USDA. Additional details of research can be found in the report for the parent CRIS 1265-32000-072-00D "Immunological and genetic basis of resistance to parasites of cattle". We have completed construction and sequence characterization of two normalized cDNA libraries (BARC 9BOV & 10BOV) derived from gut tissues undergoing parasitic infection. The libraries were constructed from the following tissues: 1) for 9BOV fundic and pyloric sections of abomasum from the O. ostertagi infected 15 week-old Holstein steers were collected three and six weeks post-infection and 2) for 10 BOV similar temporal collection of proximal jejunum was done for a C. oncophora infected group and combined with Cryptosporidium parvum infected distal ileum collected seven days after infection of two seven-day old male
Holstein calves. Normalization effectively reduced redundancy 130 and 110-fold for 9BOV and 10BOV, respectively. The normalized 9BOV and 10BOV libraries contained approximately 1.1x107 and 2.1x107 total transformants with average insert length of 1.0 and 1.2 Kb, respectively. A total of 12,672 individual colonies from each library were picked and arrayed into 33 384- well plates prior. These plates were processed in entirety for plasmid- based 5' and 3'-end (bi-directional) sequencing. Public distribution of clones and library copies is being arranged with BACPAC resources. A total of 50,112 sequence trace files were assessed for quality to generate 43,772 sequences with 100 or more high-quality bases (Phred score > 18) after trimming and removal of vector and contaminating bacterial-derived sequences. After removal of 42 EST matching O. ostertagi sequences and C. parvum accessions, the remaining 43,730 EST were deposited into GenBank. Approximately 20% of the EST represents
new bovine gene sequences relative to existing sequence information in GenBank dbEST. Analysis of the ESY information is ongoing for a manuscript. However, 20% of the clones from the libraries contain full length open reading frame cDNA inserts, and 2,000 of these have been fully sequenced. EST trace files for the 25,000 3' reads have been distributed to Texas A&M and Michigan State for design of long oligo- nucleotides for microarrays. This information has already added more than 3,000 new and annotated targets to the Michigan State array to expand their version one array above 10,000 targets.
Impacts
(N/A)
Publications
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