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Can polyA tails occur within (rather than at the end) of a sequenced tag? Consider, for example the following two sequences from NCBI: DY008075

>gi|119423037|gb|DY008075.1|DY008075 19ACACYS_UP_022_A11_29OCT2004_095 Brassica napus 19ACACYS Brassica napus cDNA 5', mRNA sequence
TGGTACGGTCAGATGCTTGCTAAAGGAGAAATAAATAGAGACATGGGTGATAGTATAAGCGGAAAGGGAA
TGATTCAGGGTGTTTCTGCAGTGGGAGCGTTTTACCAACTGCTTAGTCAGTCCAGCCTAAGTATATTGCA
TTCTGAAGAGAAGAAACCTGTGGCTCCGGTTGAATCATGTCCTATTTTGAAAACACTCTACAAGATACTC
ATCACAAGAGAACAATCAACACAAGCGATTCTGCAAGCATTAAGGGATGAAACACTGAATGACCCAAGAG
ACAGGATTGAGATTGCACAGAGCCATGCATTCTACAGGCCTTCCCTTCTAGATCAGCCTTGATTAGTCTG
TCATGGCTCATAATCCGAACTTCTAAGATCTTACTTGTGCAAACTGCAGATTCTGCTATGTTAAACATCA
TGTCTTAAAATTGATTGTTGTTCAGCCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACATGTC

or EE485195

>gi|126492146|gb|EE485195.1|EE485195 DHBN8DCT_UP_012_C05_25FEB2005_043 BRASSICA NAPUS SEEDS BNDH8DCT Brassica napus cDNA 5', mRNA sequence
GTTACAGCTGGTTGAGAACAGTGACAATTCCCGGTTGAGCAAAGAAATTGCAGACAAGAGCCACCAACTA
AGGCAAATGAGAGGAGAGGAACTTCAAGGACTTAACATAGAAGAGCTGCAACAGCTGGAAAAGGCCCTTG
AAGCTGGTTTGACGCGCGTGATTGAAACAAAGAGTGAGAAGATTATGAGTGAGATCAGTGACCTTCAAAG
AAAGGGAATGAAATTGATGGATGAGAACAAGCGGCTAAGGCAGCATGGAACACAACTAACAGAAGAGAAC
GAGCGACTAGGCAAGCAAATATATAATAATATGCATGAAAGATACGGTGGTGTTGAGTCGGAGAAGACCG
CCGTGTACGAGGAAGGGCAGTCGTCAGAGTCCATTACTAACGCCGGAAACTCCACCGGCGCTCCTGTTGA
CTCCGAGAGCTCCGATACCTCTCTTAGGCTCGGCTTACCGTATGGCGGTTAGAGATGGAACCATACAAAG
AAGTTCATGGAGTGAGGAGATGCTCTGTAGTAACAAGTGGCAATGTAGTAATTTCTCTTGTTTGATGTAA
GTTTTTGTCTGAGGAAGAGGTTTTCCTTTTATGTTCTCTTTGATATTATTATCTTTCTTCACTGCAAAAA
AAAAAAAAAAAAAAAAAAAAAAAACATGTC

It seems to me that both the polyA sequences at the end are some sort of tail rather than actual coding for poly lysines. If we BLAST either of the sequences, the polyA part doesn't align with any reliable nucleotide or protein (i.e., with the NCBI non-redundant databases). I can give more examples and show their best alignments to nr-Sequences but it will make the question too long.

Cross posted to SeqAnswers.

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As you rightly put, these poly-A sequences are not a part of the gene. I haven't done EST analysis but I generally trim poly-A tails and filter out sequences with more than 7-10 contiguous As, for RNAseq –  WYSIWYG Jul 3 '13 at 4:41
    
@WYSIWYG, but I thought they had to be at the very end. Why is there a CATGTC at the end after the polyA tail? –  highBandWidth Jul 3 '13 at 9:09
    
i tried BLASTing the second sequence against Brassica napus nucleotide collection. There is a match but only uptil 472nd residue of the query. The rest of the sequence (473-uptil the beginning of poly-A) didn't have any match in Brassica. (some gapped alignments with other non-plant genomes). Although the entry in NCBI says that the authors have removed the vector and adapter sequences, it seems that it is some kind of contaminating DNA sequence. Check their experimental procedures. –  WYSIWYG Jul 3 '13 at 10:20
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1 Answer 1

up vote 1 down vote accepted

The CATGTC sequence at the end of the poly A tail is an artefact of the method used in constructing the original cDNA library.

According to https://www.ncbi.nlm.nih.gov/nucest/EE485195.1 this EST comes from a library constructed in the Clontech vector pDNR-LIB

The Clontech SMART cDNA cloning system manuals are linked to from here and the general manual descibes the use of a primer for 1st strand synthesis:

CDS III/3' PCR Primer 5'-ATTCTAGAGGCCGAGGCGGCCGACATG-d(T)30N-1N-3'

(N = A, G, C, or T; N-1 = A, G, or C)

If you look carefully at the primer you will see that the oligo dT portion, designed to anneal to the poly-A tail of the mRNA is preceded by the sequence GACATG and a SfiI site that is used in some clever cloning strategy that I don't fully understand. What is clear however is that the use of this primer will put CATGTC immediately after the poly A in the cDNA:

           .....SfiI....
5'-ATTCTAGAGGCCGAGGCGGCCGACATGTTTTTTTTTTTTTTTTTTT...
   TAAGATCTCCGGCTCCGCCGGCTGTACAAAAAAAAAAAAAAAAAAA...5'

>>> turning bottom strand around -  5' ...AAAAAAACATGTC
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Thanks so much! That explains it. –  highBandWidth Jul 3 '13 at 13:36
    
Any idea about filtering diverse conditions on the poly-A tail? Follow up question : biology.stackexchange.com/q/9104/3922 –  highBandWidth Jul 4 '13 at 13:27
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