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MPSS: Process Overview

MPSS: Process Overview. Library to library. Run to run. Bead load to bead load. MPSS Data Structure. Replicates. Row Means. Row Variance. Merged Signature Counts. Signatures. Samples. Samples. Samples. Samples. Samples. Samples. SIGNATURE OB_COUNT GATCTGACGTTTTCTAC 56

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MPSS: Process Overview

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  1. MPSS: Process Overview Library to library Run to run Bead load to bead load

  2. MPSS Data Structure Replicates Row Means Row Variance Merged Signature Counts Signatures

  3. Samples Samples Samples Samples Samples Samples SIGNATURE OB_COUNT GATCTGACGTTTTCTAC 56 GATCACAACAGTACCAT 74 GATCAGGACACGTATCT 11 GATCCGTATGAATCTGG 1500 GATCGCTTTATGTTTTT 20 GATCCTTTGGAATCTTG 2 GATCCTGTCTCAACAAC 13 GATCAGCTTGGACAATG 24 GATCTTTGCTGGCAAGC 5 GATCCCCACCCTCCCGC1 GATCTACCTATACCCAG 67 GATCCGTgTGTGTTTAA1 GATCAGCCCATTTCTGG 4 GATCTGAATTGGCTGcA1 GATCATAACAAGAATGA 31 GATCCCGGTGTGAGGTA 1 ... MPSS MPSS MPSS MPSS MPSS MPSS Signature Digital Count Signature Digital Count Signature Digital Count Signature Digital Count Signature Digital Count Signature Digital Count Merged Signature Merged Signature Merged Signature Merged Signature Merged Signature Merged Signature Seen in other runs Single Strand Filtered Single Strand Filtered Single Strand Filtered Single Strand Filtered Single Strand Filtered Single Strand Filtered Remove Palindrome Remove Palindrome Remove Palindrome Remove Palindrome Remove Palindrome Remove Palindrome Remove Orphan Remove Orphan Remove Orphan Remove Orphan Remove Orphan Remove Orphan Remove Palindrome Not seen in other runs Filter Unreliable Filter Unreliable Filter Unreliable Filter Unreliable Filter Unreliable Filter Unreliable Normalized TPM Calculate Statistics Filter Non Significant Determine 2/4 Stepper Run Group Report Annotation Digital Signature to Expression Level SIGNATURE GATCTGACGTTTTCTAC GATCACAACAGTACCAT GATCAGGACACGTATCT GATCCGTATGAATCTGG GATCTGACGTTTTCTAC GATCGCTTTATGTTTTT GATCAGGACACGTATCT GATCCTTTGGAATCTTG GATCCGTATGTGGTTAA GATCCTGTCTCAACAAC GATCTGACGTTTTCTAC GATCACAACAGTACCAT GATCCGTATGTGgTTAA GATCACAACAGTACCAT GATCCGTATGTGGTTAA GATCTGACGTTTTCTAC ... SIGNATURE OB_COUNT GATCTGACGTTTTCTAC 56 GATCACAACAGTACCAT 74 GATCAGGACACGTATCT 11 GATCCGTATGAATCTGG 1500 GATCGCTTTATGTTTTT 20 GATCCTTTGGAATCTTG 2 GATCCTGTCTCAACAAC 13 GATCAGCTTGGACAATG 24 GATCTTTGCTGGCAAGC 5 GATCCCCACCCTCCCGC 1 GATCTACCTATACCCAG 67 GATCCGTATGTGgTTAA 1 GATCAGCCCATTTCTGG 4 GATCCGTaTGTGgTTAA 1 GATCATAACAAGAATGA 31 GATCCCGGTGTGAGGTA 1 ... SIGNATURE OB_COUNT GATCTGACGTTTTCTAC 56 GATCACAACAGTACCAT 74 GATCAGGACACGTATCT 11 GATCCGTATGAATCTGG 1500 GATCGCTTTATGTTTTT 20 GATCCTTTGGAATCTTG 2 GATCCTGTCTCAACAAC 13 GATCAGCTTGGACAATG 24 GATCTTTGCTGGCAAGC 5 GATCCCCACCCTCCCGC1 GATCTACCTATACCCAG 67 GATCCGTATGTGgTTAA 1 GATCAGCCCATTTCTGG 4 GATCGCACGTTGTACTT 5 GATCATAACAAGAATGA 31 GATCCCGGTGTGAGGTA1 ... SIGNATURE OB_COUNT GATCTGACGTTTTCTAC 56 GATCACAACAGTACCAT 74 GATCAGGACACGTATCT 11 GATCCGTATGAATCTGG 1500 GATCGCTTTATGTTTTT 20 GATCCTTTGGAATCTTG 2 GATCCTGTCTCAACAAC 13 GATCAGCTTGGACAATG 24 GATCTTTGCTGGCAAGC 5 GATCCCCACCCTCCCGC1 GATCTACCTATACCCAG 67 GATCCGTgTGTGTTTAA 1 GATCAGCCCATTTCTGG 4 GATCTGAATTGGCTGcA 1 GATCATAACAAGAATGA 31 GATCCCGGTGTGAGGTA 1 ...

  4. Samples Samples Samples Samples Samples Samples Samples Samples SIGNATURE Two Two Four Four GATCTGACGTTTTCTAC 56 55 58 54 GATCACAACAGTACCAT 74 73 74 75 GATCAGGACACGTATCT 11 12 11 10 GATCCGTATGTGGTTAA 1500 1499 1501 1498 GATCGCTTTATGTTTTT 55 0 0 0 GATCCTTTGGAATCTTG1 2 4 1 GATCCTGTCTCAACAAC 13 12 12 14 GATCAGCTTGGACAATG 24 24 25 24 GATCTTTGCTGGCAAGC 5 7 8 9 GATCTACCTATGCCCAG 67 67 66 66 GATCAGCCCATTTCTGG 4 5 7 3 GATCATAACAAGAATGA 31 30 31 29 GATCCCGGTGTGAGGTA75 70 0 0 GATCTGCCGGTGAGGTA 0 0 99 95 ... Samples Samples Samples Samples Samples Samples MPSS MPSS MPSS MPSS MPSS MPSS MPSS MPSS MPSS MPSS MPSS MPSS MPSS MPSS Signature Digital Count Signature Digital Count Signature Digital Count Signature Digital Count Signature Digital Count Signature Digital Count Signature Digital Count Signature Digital Count Signature Digital Count Signature Digital Count Signature Digital Count Signature Digital Count Signature Digital Count Signature Digital Count Merged Signature Merged Signature Merged Signature Merged Signature Merged Signature Merged Signature Merged Signature Merged Signature Merged Signature Merged Signature Merged Signature Merged Signature Merged Signature Merged Signature Single Strand Filtered Single Strand Filtered Single Strand Filtered Single Strand Filtered Single Strand Filtered Single Strand Filtered Single Strand Filtered Single Strand Filtered Single Strand Filtered Single Strand Filtered Single Strand Filtered Single Strand Filtered Single Strand Filtered Single Strand Filtered Remove Palindrome Remove Palindrome Remove Palindrome Remove Palindrome Remove Palindrome Remove Palindrome Remove Palindrome Remove Palindrome Only one run >=50 Remove Palindrome Remove Palindrome Remove Palindrome Remove Palindrome Remove Palindrome Remove Palindrome Remove Orphan Remove Orphan Remove Orphan Remove Orphan Remove Orphan Remove Orphan Remove Orphan Remove Orphan Remove Orphan Remove Orphan Remove Orphan Remove Orphan Remove Orphan Remove Orphan Filter Unreliable Filter Unreliable Filter Unreliable Filter Unreliable Filter Unreliable Filter Unreliable Filter Unreliable Filter Unreliable <=3 in all samples Filter Unreliable Filter Unreliable Filter Unreliable Filter Unreliable Filter Unreliable Filter Unreliable Normalized TPM Normalized TPM Calculate Statistics Calculate Statistics Filter Non Significant Filter Non Significant Determine 2/4 Stepper Determine 2/4 Stepper Run Group Report Run Group Report Annotation Annotation Normalized TPM Calculate Statistics Filter Non Significant Determine 2/4 Stepper Run Group Report Annotation Normalized TPM Calculate Statistics Filter Non Significant Determine 2/4 Stepper Run Group Report Annotation Normalized TPM Calculate Statistics Filter Non Significant Determine 2/4 Stepper Run Group Report Annotation Normalized TPM Calculate Statistics Filter Non Significant Determine 2/4 Stepper Run Group Report Annotation Sample 1 Sample2 SIGNATURE T_Ave T_Stdev F_Ave F_Stdev T_Ave T_Stdev F_Ave F_Stdev GATCTGACGTTTTCTAC 92 1.2 92 4.7 93 2 92 5 GATCACAACAGTACCAT 122 1.2 123 1.2 121 0 123 2.5 GATCAGGACACGTATCT 19 1.2 17 1.2 16 2.5 18 3 GATCCGTATGTGGTTAA 2489 1.2 2474 3.5 2600 50 2555 3.6 GATCGCTTTATGTTTTT 17 23.5 0 0 0 0 0 0 GATCCTTTGGAATCTTG 2 0.7 3 2.1 1 1.2 2 0 GATCCTGTCTCAACAAC 21 1.2 21 2.3 100 10 105 10 GATCAGCTTGGACAATG 40 0 40 1.2 41 0 44 1 GATCTTTGCTGGCAAGC 10 2.3 14 1.2 12 2.5 15 1.2 GATCTACCTATGCCCAG 111 0 109 0 121 0 110 0 GATCAGCCCATTTCTGG 7 1.2 8 4.7 7 1.2 8 4.7 GATCATAACAAGAATGA 51 1.2 50 2.3 60 3.2 51 2.3 GATCCCGGTGTGAGGTA 124 1.2 0 0 125 1.2 0 0 GATCTGCCGGTGAGGTA 0 0 163 0 0 0 165 0 ... Sample 1 Sample2 SIGNATURE T_Ave T_Stdev F_Ave F_Stdev T_Ave T_Stdev F_Ave F_Stdev GATCTGACGTTTTCTAC 92 1.2 92 4.7 93 2 92 5 GATCACAACAGTACCAT 122 1.2 123 1.2 121 0 123 2.5 GATCAGGACACGTATCT 19 1.2 17 1.2 16 2.5 18 3 GATCCGTATGTGGTTAA 2489 1.2 2474 3.5 2600 50 2555 3.6 GATCGCTTTATGTTTTT 17 23.5 0 0 0 0 0 0 GATCCTTTGGAATCTTG 2 0.7 3 2.1 1 1.2 2 0 GATCCTGTCTCAACAAC 21 1.2 21 2.3 100 10 105 10 GATCAGCTTGGACAATG 40 0 40 1.2 41 0 44 1 GATCTTTGCTGGCAAGC 10 2.3 14 1.2 12 2.5 15 1.2 GATCTACCTATGCCCAG 111 0 109 0 121 0 110 0 GATCAGCCCATTTCTGG 7 1.2 8 4.7 7 1.2 8 4.7 GATCATAACAAGAATGA 51 1.2 50 2.3 60 3.2 51 2.3 GATCCCGGTGTGAGGTA 124 1.2 0 0 125 1.2 0 0 GATCTGCCGGTGAGGTA 0 0 163 0 0 0 165 0 ... Sample 1 Sample2 SIGNATURE Stepper TPM Stdev TPM Stdev GATCTGACGTTTTCTAC T 92 1.2 93 2 GATCACAACAGTACCAT F 123 1.2 123 2.5 GATCAGGACACGTATCT F 19 1.2 18 3 GATCCGTATGTGGTTAA T 2489 1.2 2600 50 GATCGCTTTATGTTTTT 0 0 0 0 GATCCTTTGGAATCTTG 1 1.2 2 0 GATCCTGTCTCAACAAC F 21 1.2 105 10 GATCAGCTTGGACAATG F 40 0 44 1 GATCTTTGCTGGCAAGC F 14 1.2 15 1.2 GATCTACCTATGCCCAG T 111 0 121 0 GATCAGCCCATTTCTGG F 8 4.7 8 4.7 GATCATAACAAGAATGA T 51 1.2 60 3.2 GATCCCGGTGTGAGGTA T 124 1.2 125 1.2 GATCTGCCGGTGAGGTA F 163 0 1650 ... Sample 1 Sample2 p_value SIGNATURE TPM Stdev TPM Stdev GATCAAATTCATCTCTA 0 0 15 2 0.01014036 GATCAAATTGACCGCTT 8 6 23 9 0.07050718 GATCAAATTGGTGGGGG 11 18 2 2 0.08729055 GATCAAATTGTACTAGT 2 3 10 7 0.09091700 GATCAAATTGTGCAGTA 15 11 35 4 0.05020690 GATCCCGGTGTGAGGTA 124 1.2 125 1.2 0.58218485 GATCTGCCGGTGAGGTA 163 0 1650 0.62550128 ... SIGNATURE Two Two Four Four GATCTGACGTTTTCTAC 56 55 58 54 GATCACAACAGTACCAT 74 73 74 75 GATCAGGACACGTATCT 11 12 11 10 GATCCGTATGTGGTTAA 1500 1499 1501 1498 GATCGCTTTATGTTTTT 55 0 0 0 GATCCTTTGGAATCTTG 1 2 4 1 GATCCTGTCTCAACAAC 13 12 12 14 GATCAGCTTGGACAATG 24 24 25 24 GATCTTTGCTGGCAAGC 5 7 8 9 GATCTACCTATGCCCAG 67 67 66 66 GATCAGCCCATTTCTGG 4 5 7 3 GATCATAACAAGAATGA 31 30 31 29 GATCCCGGTGTGAGGTA75 74 0 0 GATCTGCCGGTGAGGTA 0 0 99 99 ... Run Group Report

  5. Calculate Mean, Tpm, and Standard Deviation Calculate mean and tpm of 2-stepper runs for signature 1 Weighted Average = (20 + 0 + 17) / (671873 + 704108 + 727162) = 1.75927e-5 Average Clone Counts Normalize Factor = (671873 + 704108 + 727162) / 3 = 701047.6 Tpm Normalize Factor = 1e6 Mean = Weighted Average * Ave. Clone Counts Normalize Factor = (1.75927e-5) * (701047.6) = 12.333 = 12 Tpm = Weighted Average * tpm Normalize Factor = (1.75927e-5) * (1e6) = 17.592 = 18 • Repeat to get mean and tpm of 4-stepper runs for signature 1 • Repeat for all signatures in RunGroup • Calculate standard deviation as well, normalized to 1 million

  6. Lynx MPSS Statistic Model The data produced with MPSS are of the “categorical" type from a statistical point of view. Therefore, a test of significance for differential expression between samples can be carried out using a normal approximation test (also referred to as Z-test). If x1 and x2 represent the abundance of a specific signature in samples 1 and 2, the proportions thus follow a binomial distribution. Because n1and n2 are large in MPSS (typically in the order defined by equation (1) where the unknown parameters p and q can be estimated as and ,respectively. and (1) Thus, the test statistics defined by equation (2) below follow a standard normal distribution. Equation (2) is a two-tailed, normal approximation test for binomial proportions. The digital nature of MPSS allows one to use equation (2) to calculate the percentage difference in expression that is statistically significant between multiple samples. These characteristics are in contrast to the analyses for gene expression data generated by hybridization based methods, such as microarrays, where a significance test is possible only if the experiment is replicated many times, and where differential expression can usually be detected only for genes with relatively high levels of expression and with a large difference between samples. (2)

  7. Calculate P-values (where x is observed count, and c is clone count) p = 0.2316419 b1 = 0.319381530 b2 = - 0.356563782 b3 = 1.781477937 b4 = - 1.821255978 b5 = 1.330274429

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