1. EST Sequence Cleaning and Quality Control FW4089 Bioinformatics H. Jiang

2. Background Expressed Sequence Tag (EST): small pieces of DNA seq (300-500bp) sequenced at one or both ends of an expressed gene. Advantage: fast and easy, to fish a gene out. Challenge: seq errors, redundant ESTs,.. ~12, 000 from 8 cDNA libraries: Control Apex (CA), Transgenic Apex (TA) Control Apex (CA), Transgenic Apex (TA) Control Apex (CL), Transgenic Apex (TL) Control Apex (CL), Transgenic Apex (TL) Control Apex (CR), Transgenic Apex (TR) Control Apex (CR), Transgenic Apex (TR) Control Apex (CS), Transgenic Apex (TS) Control Apex (CS), Transgenic Apex (TS)

3. Sequence Cleaning (1) Base-calling program---Phred: Input: chromatogram files Input: chromatogram files Output: seq file and quality file (scores) Output: seq file and quality file (scores) >phred –id./passTrace –sd./passSeq/ -qd./passQual/ DNA sequence cleaning: quality trimming and vector removal---Lucy: Lucy Steps: Read input seq#, seq info, and quality info Read input seq#, seq info, and quality info Chop off splice sites Chop off splice sites Remove vector insert Remove vector insert Produce output seq for fragment assembly. Produce output seq for fragment assembly.

4. Sequence Cleaning (2) Lucy Input: seq file and quality file >lucy [parameters] seqFile qualFile [2ndSeqFile] Lucy major parametersto set up: -vector vector_completeSeq splice_site_file (splice_site_file: 2 splice-site seq before and after the insertion point on the vector) splice.begin(150 bp) ____________________________________________________________________________ M13 Rev. Primer EcoRI Linker 1 Linker 4 EcoRI splice.end(150 bp) ____________________________________________________________________________ half of Linker 1 Linker 4 EcoRIM13 For Primer Lucy Output: identified locations of good/clean region identified locations of good/clean region trim seq without vector, linker, Ns (<3% Ns), and poly A/Ts. trim seq without vector, linker, Ns (<3% Ns), and poly A/Ts. Other check-up (e.g. chloroplast, mitochondrial, …) Submit 11, 545 ESTs to GenBank

5. Any Problem? An example. In-house re-sequence clone at plate position of NP9A.A1, its seq should match MTU2CA.P16.F09, which was submitted. However, blast ESTdb at NCBI did not find any match with our seq. Problems? Half of the new seq is vector contamination Half of the new seq is vector contamination Seq cleaning problems? Seq cleaning problems? ClustalW-multiple seq alignment of new seq, Lucy- trimmed seq and Seq from seq company. ClustalW-multiple seq alignment of new seq, Lucy- trimmed seq and Seq from seq company.

6. NP_9A_A1_new_ AGAATACGCCAGCTTGGTACCGAGCTCGGATCCCTAGTAACGGCCGCCAG Lucy2CA.P16.F09_299bp_ -------------------------------------------------- Larkun-trimmed_751bp_ ---------------------------------CCTNGNACGGCCGC-AG NP_9A_A1_new_ TGTGCTGGAATTCGCCCTTTCGAGCGGCCGCCCGGGCAGGTCAACGGTTG Lucy2CA.P16.F09_299bp_ ----------------------------------------GCAACGGTTG Larkun-trimmed_751bp_ TGTGCTGGAATTCGCCCTTTCGAGCGGCCGCCCGGGCAGGNNAACGGTTG ******** NP_9A_A1_new_ ACTTCAATCCAACCATAATTAAACGGCGATAGATCATAATTTCAGTCAAG Lucy2CA.P16.F09_299bp_ ACTTCAATCCAACCATAATTAAACGGCGATAGATCATAATTTCAGTCAAG Larkun-trimmed_751bp_ ACTTCAATCCAACCATAATTAAACGGCGATAGATCATAATTTCAGTCAAG ************************************************** NP_9A_A1_new_ TTCTAAGAACCCATTATCAAATTATTATCCAACAACAACAATAATAATTT Lucy2CA.P16.F09_299bp_ TTCTAAGAACCCATTATCAAATTATTATCCAACAACAACAATAATAATTT Larkun-trimmed_751bp_ TTCTAAGAACCCATTATCAAATTATTATCCAACAACAACAATAATAATTT ************************************************** NP_9A_A1_new_ CTCATTCGAAGAGAATCGTAGAATTCATAATCTAAATCGAAAAAAAAACT Lucy2CA.P16.F09_299bp_ CTCATTCGAAGAGAATCGTAGAATTCATAATCTAAATCGAGGGGGAAACT Larkun-trimmed_751bp_ CTCATTCGAAGAGAATCGTAGAATTCATAATCTAAATCGNNNGGNNAACT *************************************** **** NP_9A_A1_new_ AAAAATCCATCAAATTAAACAAAAACAAACCCGAAGATGGATGAATCAAT Lucy2CA.P16.F09_299bp_ AAAAATCCATCAAATTAAACAAAAACAAACCCGAAGATGGATGAATCAAT Larkun-trimmed_751bp_ AAAAATCCATCAAATTAAACAAAAACAAACCCGAAGATGGATGAATCAAT ************************************************** NP_9A_A1_new_ TGGTCTTGGGATCAGGAAGGGGGAGAAAACCAGNACCAGGAGCAGAACCA Lucy2CA.P16.F09_299bp_ TGGTCTTGGGATCAGGAAGGGGGAGAAAACCAGCACCAGGAGCAGAACCA Larkun-trimmed_751bp_ TGGTCTTGGGATCAGGAAGGGGGAGAAAACCAGCACCAGGAGCAGAACCA ********************************* **************** NP_9A_A1_new_ CGAGGCGGAGAAAACCCAAGAAACTTCTGCAAATTAGTGCCTCGGCCGCG Lucy2CA.P16.F09_299bp_ CGAGGCGGAGAAAACCCAAGAAACTTCTGCAAATTAGTG----------- Larkun-trimmed_751bp_ CGAGGCGGAGAAAACCCAAGAAACTTCTGCAAATTAGTGCCTCGGCCGCG *************************************** NP_9A_A1_new_ ACCACGCTAAGGGCGAATTCTGCAGATATCCATCACACTGGCGGCCGCTC Lucy2CA.P16.F09_299bp_ -------------------------------------------------- Larkun-trimmed_751bp_ ACCACGCTAAGGGCGAATTCTGCAGATATCCATCACACTGGCGGCCGCTC linker seq Seq difference EcoRI

7. Find Questionable Sequences Strategy: Find and compare sequences of ‘clean region’ defined by Lucy (coordinates are in debug files produced by Lucy) Find and compare sequences of ‘clean region’ defined by Lucy (coordinates are in debug files produced by Lucy) Find those seq that have low quality scores and different between Phred base calls and Lark calls Find those seq that have low quality scores and different between Phred base calls and Lark calls Criteria: minimum length >100, >10 continuous low quality scores ( 100, >10 continuous low quality scores (<16) Compare difference between Lark and Lucy seq Compare difference between Lark and Lucy seq Manually check some chromatogram files Manually check some chromatogram files

8. Program 1 From debug file get coordinate file: Where good/clean region starts and ends Where good/clean region starts and ends Program 1: (a C program) Usage:./clean coordfile qualfile threshhold regionlength [outputfile] 1. get coordfile: ESTID+CLR 2. qualfile: quality file 3. threshold: 16 (Lucy default) 4. regionlength: 10 (10 continuous low scores) 5. optional output filename.

9. Program 2 Program 2: (a unix-based Flex program) Partial code: 1. Define rules (patterns): e.g., CHAR [A-Z]; INT -?[0-9]; MTUID; BAD {INT}* 1. Define rules (patterns): e.g., CHAR [A-Z]; INT -?[0-9]; MTUID; BAD {INT}* 2. Action 2. Action {BAD}{ if ( ? <=16 ) if ( ? <=16 ){ ++count; ++count;} if (count >= 10 && inbadregion = 0) { strncp (?)? strncp (?)? printf("\n%s ", yytext); printf("\n%s ", yytext);}else{ count = 0; count = 0; inbadregion =1; inbadregion =1;} 3. main() 3. main() 4. Compile and produce an executable file. 4. Compile and produce an executable file.

10. Result (1) By C and Flex program, found ~2400 seq had 10 consecutive low scores Lucy claimed that average probability of error (score<=16) over the final clean range is 2.5%. Next: compare base calls between Lucy trimmed and Lark sequences. By 2 methods: GAP and Local Blast

11. GAP of 2400 seq pairs (1) Convert file format: gcg>fromfasta bigfastaFile of Lark and Lucy Run a shell script: gap Lucyseq1 Larkseq1 (all 2400 pairs) Output : *.pair file (example see below) GAP of: mtu2ta.p13.h06.seq check: 4559 from: 1 to: 358 MTU2TA.P13.H06 to: origmtu2ta.p13.h06.seq check: 438 from: 1 to: 358 origMTU2TA.P13.H06 Gap Weight: 50 Average Match: 10.000 Length Weight: 3 Average Mismatch: 0.000 Quality: 3248 Length: 369 Ratio: 9.073 Gaps: 4 Percent Similarity: 99.712 Percent Identity: 97.983

12. GAP of 2400 seq pairs (2) Perl program to parse *.pair output Use regular expression to find key words and extract the corresponding values: if ($line=~/Ratio: /) { $line =~s/Gaps/:Gaps/; @crx =split (":",$line); $crx[1]=~s/^\s*//; $crx[1]=~s/\s*$//; $crx[3]=~s/^\s*//; $crx[3]=~s/\s*$//; $ratio =$crx[1]; $gaps =$crx[3]; Criteria: min length >100, gap>=1, identity 100, gap>=1, identity <95%. Found: total 188 seq different. Found: total 188 seq different.

13. Local Blast of 2400 seq pairs (1) Format Lark orig seq to database files >formatdb –i orig –p F –o T Blast Lucy trimmed seq (one bigfastaFile) with orig >blastall –i lucy –d orig –p blastn –e 0.05 –m 9 –b 1 >blastout (output redireciton) Blast output: # BLASTN 2.2.4 [Aug-26-2002] # Query: MTU2CA.P10.A07 # Database: orig # Fields: Query id, Subject id, % identity, alignment length, mismatches, gap openings, q. start, q. end, s. start, s. end, e-value, bit score MTU2CA.P10.A07 MTU2CA.P10.A07 99.83 573 1 0 1 573 1 573 0.0 1130.4 MTU2CA.P10.A07 MTU2CA.P4.E01 99.48 5733 0 1 573 1 573 0.0 1114.6 MTU2CA.P10.A07 MTU2CA.P4.A02 99.07 5405 0 34 573 6 545 0.0 1033.3

14. Local Blast of 2400 seq pairs (2) Cut-off criteria: Cut-off criteria: pick the top hit(same IDs), select those with identity 100, and gap>=1 Parse the output by a Perl program Parse the output by a Perl program Found total 158 sequences. Found total 158 sequences.

15. Compare GAP and Blast Results Compare results: Blast is quick and not accurate (e.g. unknown reason to open a gap for a perfect matched seq pair). Compare results: Blast is quick and not accurate (e.g. unknown reason to open a gap for a perfect matched seq pair). Find common seq IDs: Find common seq IDs: total of 144 seq (1.2% of 11, 545 submitted seq). total of 144 seq (1.2% of 11, 545 submitted seq). Manual check on their chromatograms Manual check on their chromatograms (To be continued……)

16. A chromatogram example NNNGGNN